1: Mol Pharmacol. 2005 Aug;68(2):372-82. Epub 2005 May 20. Chemotherapy compounds in cervical cancer cells primed by reconstitution of p53 function after short interfering RNA-mediated degradation of human papillomavirus 18 E6 mRNA: opposite effect of siRNA in combination with different drugs. Koivusalo R, Krausz E, Helenius H, Hietanen S. Dept. of Obst and Gynecology, Turku University Central Hospital, Kiinamyllynkatu 4-8, 20520 Turku, Finland. Constant expression of E6 and E7 mRNA by high-risk human papillomaviruses (HPV) abrogates p53 and retinoblastoma protein function, respectively, and is essential for the development of cervical cancer. Despite E6, some chemotherapy drugs can stabilize p53 in cervical cancer cells. It is not known how chemotherapy-induced p53 activation and cytotoxicity are affected when the amount of E6 mRNA is decreased before the drug treatment. In this study, HPV18-positive HeLa cervical cancer cells were transfected with short interfering RNA (siRNA) molecules targeting HPV18 E6 mRNA before treatment with carboplatin, cisplatin, doxorubicin, etoposide, gemcitabine, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, and topotecan. Transfection with siRNA was followed by nuclear accumulation of p53, but the effect was transient despite continuously suppressed HPV mRNA levels. When treatment with E6 siRNA was coupled with chemotherapy, the p53 activity after treatment with carboplatin and paclitaxel was additively increased, whereas the p53 activation induced by the rest of the drugs was synergistically increased. Treatment with E6 siRNA alone moderately inhibited HeLa cell proliferation but did not induce detectable apoptosis. The combined cytotoxic effect of E6 siRNA and chemotherapy ranged from subadditive to synergistic, depending on the drug. The decrease of E6 mRNA sensitized HeLa cells, for example, to doxorubicin and gemcitabine but counteracted the cytotoxicity of cisplatin and etoposide. In conclusion, activating p53 by degrading E6 mRNA may either increase or decrease the chemosensitivity of cervical cancer cells, depending on the chemotherapy compound. PMID: 15908516 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 2: Indian J Dent Res. 2003 Oct-Dec;14(4):214-9. p53 aberrations in oral sub mucous fibrosis and oral cancer detected by immunohistochemistry. Bathi RJ, Prabhat. Oral Medicine Diagnosis And Radiology, SDM College of Dental Sciences And Hospital, Dharwad-09. sdmtl@.sanchar.net.in Study of expression of p53 oncoprotein in several precancerous and cancer have been done, but only one literature is available regarding p53 expression in Oral Sub Mucous Fibrosis (OMSF), hence this study was taken up (i) to determine the expression of aberrant p53 in Oral Sub Mucous Fibrosis (OSMF) and Oral Squamous cell carcinoma (SCC) patients. (ii)To study correlation if any between p53 expression and degree of dysplasia in OSMF and SCC patients and (iii)To study correlation if any between p53 expression and habits in OSMF and SCC patients. Study Design consists of biopsy specimens of 38 cases of OSMF and 37 cases of Squamous cell carcinoma were subjected for staining by immunohistochemistry for p53 protein using LSAB visualization system kit. Clinical details along with habits were recorded and the data analyzed with t- test and chi- square test. Results of the study reveals 18 cases of OSMF and 26 cases of SCC were positive for p53 protein. Only 4 cases of SCC showed (++)grade and the rest all had (+)grade. Out of 75 patients, 65 had the habit of smoking and chewing, 4 patients history of habit was not known. Among patients with habits (65), 40 specimens were +ve for p53 stain and 2 out of 6 without history of habit, 2 out of 4 unknown history of habit took up p53 stain. To conclude study showed higher percentage of p53 positive cells in oral cancer cases when compared to oral sub mucous fibrosis cases. It suggests p53 expression may correlate with increase in dysplasia or malignant transformation. Both smoking and chewing habits had a significant role in p53 positive expression. PMID: 15328987 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Ai Zheng. 2004 Mar;23(3):235-42. Circadian expression of dihydropyrimidine dehydrogenase, thymidylate synthase, c-myc and p53 mRNA in mouse liver tissue. Wu MW, Xian LJ, Li XM, Pasquale I, Francis L. INSERM E 0354 Chronotherape utique des cancers, Hopital Paul Brousse and Universite Paris XI, 94807 Villejuif Cedex, France. BACKGROUND & OBJECTIVE: Anti-cancer effect of 5-Fluorouracil (5-FU) is mediated mainly by inhibition of the thymidylate synthase (TS), while dihydropyrimidine dehydrogenase (DPD) is an initial and a rate-limiting catabolic enzyme of 5-FU. In this study, the mRNA expression profiles of TS, DPD, p53 and c-myc were investigated in mouse liver. METHODS: A total of 24 male B6D2F1 mice were involved in this study. All the mice were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD12:12) for 4 weeks. Body temperature and rest-activity were monitored with an intra-peritoneal sensor. All the mice were sacrificed at 3, 7, 11, 15, 19, 23 HALO (hours after light onset) respectively and liver samples were obtained and immediately frozen in liquid nitrogen. Total RNA was extracted from the frozen liver samples and one-step real-time quantitive RT-PCR was performed using LightCycler - RNA Amplification Kit SYBR Green I system. RESULTS: Both body temperature and rest-activity displayed similarly rhythmic patterns with peak times located in darkness, while the trough time was located in the light span. DPD showed a circadian expression in mRNA level with a peak at about 16 HALO (P=0.0012). TS showed a trend for a circadian rhythm, with a peak during light (P=0.079). Neither c-myc nor p53 displayed significant circadian rhythm. CONCLUSION: The 24-h patterns in DPD and TS expression were approximately 12 h out of phase, supporting a coordinated regulation of both transcriptional pathways relevant for 5-FU chronopharmacology. PMID: 15025949 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Oncogene. 2004 Mar 18;23(12):2177-87. Deacetylase recruitment by the C/H3 domain of the acetyltransferase p300. Simone C, Stiegler P, Forcales SV, Bagella L, De Luca A, Sartorelli V, Giordano A, Puri PL. Laboratory of Gene Expression, Dulbecco Telethon Institute (DTI) at Fondazione A Cesalpino, University of Roma La Sapienza, Italy. The balance between acetylation and deacetylation of histone and nonhistone proteins controls gene expression in a variety of cellular processes, with transcription being activated by acetyltransferases and silenced by deacetylases. We report here the formation and enzymatic characterization of a complex between the acetyltransferase p300 and histone deacetylases. The C/H3 region of p300 was found to co-purify deacetylase activity from nuclear cell extracts. A prototype of class I histone deacetylases, HDAC1, interacts with p300 C/H3 domain in vitro and in vivo. The p300-binding protein E1A competes with HDAC1 for C/H3 binding; and, like E1A, HDAC1 overexpression interferes with either activation of Gal4p300 fusion protein or p300-dependent co-activation of two C/H3-binding proteins, MyoD and p53. The exposure to deacetylase inhibitors could reverse the dominant-negative effect of a C/H3 fragment insulated from the rest of the molecule, on MyoD- and p53-dependent transcription, whereas inhibition by E1A was resistant to trichostatin A. These data support the hypothesis that association between acetyltransferases and deacetylases can control the expression of genes implicated in cellular growth and differentiation, and suggest that the dominant-negative effect of the p300 C/H3 fragment relies on deacetylase recruitment. PMID: 14968110 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Histol Histopathol. 2004 Jan;19(1):9-14. Expression and significance of cell immunohistochemical markers (HHF-35, CD-31, Bcl-2, P-53 and apopDETEC) in hypertrophic cardiomyopathy. Martinez-Diaz F, Bernal-Gilar M, Gomez-Zapata M, Luna A. Department of Pathology, Medicine School of Murcia, University of Murcia, Murcia, Spain. There are several hypotheses concerning the pathogenesis of hypertrophic cardiomyopathy (genetic, ischaemic, immune, inflammatory and apoptosis induction). We have studied three types of cardiomyopathy in order to observe the expression and assess the significance of different immunohistochemical markers (muscular actin, CD-31, proliferation cell nuclear antigen -PCNA-, Ki-67, and markers related with programmed cell death, bcl-2, p-53 and apopDETEC). We studied different microscopic (haematoxylin-eosin and Masson's thrichrome) and immunohistochemical parameters (streptavidin-biotin-peroxidase and "in situ" hybridisation) of forty cases: ten each of hypertensive hypertrophic cardiomyopathy, essential hypertrophic cardiomyopathy, hypertrophic cardiomyopathy in patients treated with chemotherapy and morphologically "normal" hearts. Our findings point to an absence of structural marker expression (actin and CD-31) in cases of hypoxic damage. The distribution and intensity of apoptosis markers, a seen by "in situ" hybridisation were irregular, and the rest of the markers studied showed negative results, with the exception of acridin orange (a marker of hypoxic damage). In our opinion, the above immunohistochemical markers, especially actin and CD-31, could be used for differentiating hypoxic lesions in these three types of cardiomyopathy. Moreover, it is difficult to know the significance of the apoptosis markers, because the autolysis process produces cross reactions with false positive results. We think that there is a need for new studies on DNA breakdown processes during the post-mortem interval. To avoid autolysis problems the post-mortem material needs to be as fresh as possible. PMID: 14702165 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Cancer Genet Cytogenet. 2004 Jan 1;148(1):66-70. Chromosome analyses of 16 cases of Wilms tumor: different pattern in unfavorable histology. Peres EM, Savasan S, Cushing B, Abella S, Mohamed AN. Division of Pediatric Hematology/Oncology, Children's Hospital of Michigan, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA. Cytogenetic analyses of 16 cases of Wilms tumor with abnormal karyotypes were reviewed, 15 cases of unilateral tumor and 1 bilateral. Three tumors exhibited an unfavorable histology (i.e., anaplastic changes); the rest fell into the favorable histology group. Of the 17 tumors with abnormal clonal aberrations, 9 tumors were hyperdiploid (53%), 7 had pseudodiploid karyotypes (41%), and 1 was hypodiploid (6%). The most common numerical aberrations in descending order of frequency were gain of chromosomes 12, 8, and 6 and loss of chromosome 16. Structural rearrangements mostly involved chromosome 1, followed by chromosomes 7, 14, and 17. Clustering of breaks around 1p22 approximately p31-->pter resulting in partial loss of 1p was the most frequent structural aberration. Additionally, i(7q) was observed as a sole abnormality in two tumors and a 7p translocation in two other tumors. Two other recurrent abnormalities were a partial deletion of 14q, seen in three tumors, and complete loss of chromosome 14 in one tumor. All three Wilms tumors with unfavorable histology had abnormalities of 17p, resulting in TP53 gene deletion. These findings provide further support for the importance of gains of chromosomes 12, 8, and 6 and loss of 1p in the development of Wilms tumor. The results also support the association of unfavorable-histology Wilms tumors with TP53 deletion. The nonrandom losses of 16/16q, 7p, and 14q may point to the importance of genomic imbalance in the pathogenetic consequences and progression of Wilms tumor. PMID: 14697643 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Sichuan Da Xue Xue Bao Yi Xue Ban. 2003 Jul;34(3):559-61. [Research on motor dysfunction and the role of CTP after traumatic brain injury in rats] [Article in Chinese] Zhao J, Liu Q, Cui J, Hong J, Song Z. Department of Fundamental Medicine, Northern China Coal Medical College, Tanshan 063000, China. OBJECTIVE: This is a study on the level of molecular biology to clarify the possible mechanism of delayed neural death after traumatic brain injury (TBI) and to probe into the influences of motor function of cytidine triphosphate(CTP) on rats after brain trauma. METHOD: The model of severe closed traumatic brain injury (TBI) was established according to the method created by Marmarou in 1994. 300 Wistar rats were divided randomly into TBI group (n = 96), CTP treating group and sham operation group, and each of these three groups was divided into 8 subgroups, namely, 3, 6, 12, 24, 48, 72, 168 and 336 hours. At the same time, the rest 12 rats were taken as the normal group for comparison. The CTP treating group after injury were treated with injection of CTP (30 mg/(kg.d)) in peritoneum; the TBI group, the sham operation group and the normal group were treated with normal saline for comparison at the same time and all were killed at each time point. Another 48 rats were divided randomly into 4 groups, namely, brain injury group, CTP treating group, sham operation group and the normal group, and their motor dysfuction was evaluated using Neurological Severity Score (NSS). The methods such as immunohistochemistry, TUNEL and NSS were used to dynamically observe the pathological changes in cortex of rats, the expression of P53 protein and the motor function after TBI. RESULTS: The increased expression of P53 protein and neural apoptosis showed up in cortex and motor dysfunction occurred after TBI. CONCLUSION: The increased expression of P53 protein may bring about nerve cell apoptosis after TBI. Motor dysfunction is the inevitable outcome of nerve cell apoptosis in cortex. CTP can reduce P53 protein expression and nerve cell apoptosis, and it can improve motor function. PMID: 12910721 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Zhonghua Er Bi Yan Hou Ke Za Zhi. 2000 Oct;35(5):342-4. [Expression of simian virus 40 large T-antigen and p53 protein in chinchilla middle ear epithelial cell line and its significance] [Article in Chinese] Jin S, Jin Y, Cui Z. Department of Otorhynolaryngology, Medical College Hospital, Yanbian University, Yanji, Jilin 133000, China. OBJECTIVE: To identify the expression of simian virus 40 large T-antigen (SV40TAg) and p53 protein in the cell nuclei of chinchilla middle ear epithelial cell line and explore the significance of the expression. METHODS: To examine the expression of SV40TAg and p53 protein in cell nuclei of chinchilla middle ear epithelial cell which had been infected and had not been infected with Ad 12-SV40 hybrid virus by immunocytochemistry method. RESULTS: All of the cell nuclei in 1 of 8 culturing dishes which infected with the Ad12-SV40 virus expressed the SV40TAg and p53 protein and had cultured over 46 passages. They have become a cell line. All of the cells in rest 7 culturing dishes and the control cells did not express the SV40TAg and p53 protein in their nuclei and died after culturing 6-8 passages. CONCLUSION: The SV40TAg and p53 protein play some important role in process of cell immortalization. The expression of SV40TAg and p53 protein in cell nuclei of CMEE-1 cell line has close interrelations with establishing the CMEE-1 cell line. PMID: 12768732 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 9: Di Yi Jun Yi Da Xue Xue Bao. 2002 Apr;22(4):323-4. p53 expression and cancer cell invasion in the distal bowel wall of patients with rectal carcinoma. Deng HJ, Li GX, Qing SH, Huang XC. Department of General Surgery, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. OBJECTIVE: To determine an adequate distal bowel length to be excised for safe surgical removal of rectal carcinoma. METHODS: p53 expression and patterns of cancer cell invasion into the bowel wall distal to the tumors were studied in 68 surgically removed rectal carcinoma specimens with immunohistochemical and routine pathological methods respectively. RESULTS: In 52.9% (36/68) of the cases, cancer cell infiltration in the bowel was found microscopically, among which 32 (88.9%) had the infiltration confined within 2 cm from the primary tumors, with the rest cases within 3 cm. The scope of the distal infiltration of the cancer cells was correlated to the gross morphology, histological type and Dukes' stages of the primary tumors. Of the 16 cases (23.5%) positive for p53 expression in the distal bowel mucosa, 13 had p53 expression profile detected within 2 cm, all within 3 cm. The expression of p53 in the distal bowel mucosa was not correlated with the clinical and pathological features of the primary rectal carcinoma. CONCLUSION: Excision till the compromised bowel 3 cm distal to the tumor may ensure safe removal of rectal cancer. PMID: 12390734 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 10: J Mol Biol. 2002 Mar 22;317(2):279-90. A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin. Maler L, Sastry M, Chazin WJ. Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, Sweden. Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site. Copyright 2002 Elsevier Science Ltd. PMID: 11902843 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Zhonghua Bing Li Xue Za Zhi. 2001 Apr;30(2):85-8. [Gene rearrangement and p53 expression in defining the nature of angioimmunoblastic lymphadenopathy] [Article in Chinese] Zhao P, Ji X, Zhang H, Jiang T, Sun X. Department of Pathology, The Hospital of Qingdao University Medical College, Qingdao 266003, Shandong Province, China. OBJECTIVE: To investigate gene rearrangement and p53 expression in defining the nature of angioimmunoblastic lymphadenopathy. METHODS: DNA was extracted from paraffin-embedded tissue blocks of 44 angioimmunoblastic lymphadenopathy (AIL) patients and analyzed with polymerase chain reaction (PCR) for IgH and TCRgamma gene rearrangement. Immunohistochemistry staining was used to detect p53 protein expression. Thirty-five cases were followed-up. RESULTS: 12 out of 44 cases (27.3%) showed TCRgamma gene rearrangement and 2 (4.5%) showed IgH gene rearrangement. Rearrangement of both IgH and TCRgamma genes were detected in 2 cases (4.5%). 14 cases (31.8%) showed p53 positive expression, among which 12 showed positive rearrangement and 2 showed negative (P < 0.01). Eight out of 11 patients of positive gene rearrangement died in one year, while only 3 patients were still alive at the eighteenth month of follow-up, three of 24 patients of negative gene rearrangement were found dead at the time of the one year follow-up, while the rest 21 patients were alive and the longest survival time was 96 months. CONCLUSIONS: Gene rearrangement can define the pathological nature of AIL. The expression of p53 is highly related to gene rearrangement, and thus an important immunological marker in research on AIL. PMID: 11866959 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Acquir Immune Defic Syndr. 2002 Feb 1;29(2):181-3. Lymphoid neoplasms in HIV-positive individuals in India. Agarwal B, Ramanathan U, Lokeshwas N, Nair R, Gopal R, Bhatia K, Naresh KN. Lymphoma Registry, Department of Pathology, Tata Memorial Hospital, Mumbai, India. The HIV epidemic in the Asian subcontinent has a significant impact on India. Patients with AIDS have an increased risk of developing non-Hodgkin lymphoma (NHL). In this study, we have investigated the pattern of distribution of lymphoid neoplasms and also studied the Epstein-Barr virus (EBV)-association and p53 expression in 35 HIV-positive patients from India. The biopsy samples were studied for histology and for expression of CD20, CD3, CD15, CD30, light chains, CD138, bcl-6, epithelial membrane antigen, EBV-latent membrane protein-1, and p53 protein. In situ hybridization was performed with digoxigenin-labeled anti-sense EBV-encoded nuclear RNA-1 (EBER-1) probe. Polymerase chain reaction (PCR) was performed on DNA extracted from paraffin sections for EBV-subtype analysis. The 35 cases included 7 cases of Hodgkin disease (HD), 4 cases of plasmacytoma (PL), and 24 cases of NHL. Among the cases of NHL, 3 were Burkitt lymphoma (BL), 4 were diffuse large B-cell lymphoma (DLBL) of centroblastic type (CBL), 10 were DLBL of immunoblastic type (IBL), 4 were high-grade B-cell lymphoma (unspecified) and the rest were other subtypes. EBV-association was noted in all cases of HD, 2 of 3 BL, and 3 of 10 IBL. PCR analysis of the EBNA-3C gene revealed amplimers corresponding to type A. A p53 protein overexpression was noted in 6 of 10 IBLs, 1 of 3 BLs, 2 of 3 CBLs, and 5 of 7 cases of HD. This is the first reported study of lymphoid malignancies in HIV-positive individuals from India. PMID: 11832689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Folia Histochem Cytobiol. 2001;39 Suppl 2:81-3. Effect of tyrphostins on programmed cell death in colon adenocarcinoma cell line LS-180. Dudzisz-Sledz M, Mizerski G, Marzec B, Korszen-Pilecka I, Rudzki S, Wojcierowski J, Mandziuk S. Department of Human Genetics, Medical University, Lublin, Poland. mdudzisz@yahoo.com Programmed cell death is an important process in the regulation of cellular proliferation, rest, differentiation and death. It is a genetically controlled process with characteristic biochemical and morphological features. Apoptosis directly regulates tumorigenesis and its induction could be a useful method of cancer therapy. Cancer cells could be influenced by some factors which induce apoptosis. We investigated the influence of tyrphostins, that specifically inhibits protein tyrosine kinases and stops the cell cycle in apoptosis of the colon adenocarcinoma cell line LS180. We used them at the concentration of 1-10 microM for 24 and 48 hours. We detected apoptosis using techniques that monitor either biochemical and morphological features of this process, such as staining with 7-amino-actinomycin D, staining with Grunwald-Giemsa, TUNEL reaction, in situ hybridization and with immunoperoxidase staining procedures. We examined the expression of genes and proteins connected with programmed cell death (p53, c-myc, p21, bcl-2). We estimated the results by cytophotometry and documented them by colour photography. We found that tyrphostin rapidly inhibits the cell cycle, particularly at the concentration of 5 microM. The expression of genes and proteins was strongly correlated with the increased apoptotic cell death conforming to the results of TUNEL and staining methods. PMID: 11820638 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: Eur J Neurosci. 2001 Oct;14(8):1303-12. Biological activity of RE-1 silencing transcription factor (REST) towards distinct transcriptional activators. Lietz M, Bach K, Thiel G. Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, D-66421 Homburg, Germany. The zinc finger protein RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal genes in non-neuronal tissues. We have analyzed the ability of REST and the REST mutants, RESTDeltaN and RESTDeltaC lacking either the N-terminal or C-terminal repression domains of REST, to inhibit transcription mediated by distinct transcriptional activator proteins. For this purpose we have designed an activator specific assay where transcription is activated as a result of only one distinct activation domain. In addition, binding sites for REST were inserted in the 5'-untranslated region or at a distant position downstream of the polyadenylation signal. The results show that REST or the REST mutants containing only one repression domain were able to block transcriptional activation mediated by the transcriptional activation domains derived from p53, AP2, Egr-1, and GAL4. Moreover, REST, as well as the REST mutants, blocked the activity of the phosphorylation-dependent activation domain of Elk1. However, the activity of the activation domain derived from cAMP response element binding protein 2 (CREB2), was not inhibited by REST, RESTDeltaN or RESTDeltaC, suggesting that REST is able to distinguish between distinct transcriptional activation domains. Additionally, the activator specific assay, together with a positive-dominant mutant of REST that activated instead of repressed transcription, was used in titration experiments to show that REST has transcriptional repression and no transcriptional activation properties when bound to the 5'-untranslated region of a gene. PMID: 11703459 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Eur J Surg Oncol. 2001 Sep;27(6):574-80. Prognostic significance of p53, bax and bcl-2 gene expression in patients with laryngeal carcinoma. Georgiou A, Gomatos IP, Ferekidis E, Syrigos K, Bistola V, Giotakis J, Adamopoulos G, Androulakis G. Department of Otolaryngology, Hippokration Hospital, Athens, Greece. AIM: This study was designed to examine the prognostic significance of the coexpression of three genes (bax, bcl-2 and p53) which play a critical role in the apoptotic mechanisms in patients with squamous cell laryngeal carcinoma. MATERIALS AND METHODS: The immunohistochemical expression of bcl-2, bax and p53 genes was retrospectively examined in 38 patients with squamous cell laryngeal carcinoma and in five controls (necrotomic tissue). Tissue specimens were obtained both during the diagnostic biopsy and at the time of surgery. Clinicopathological and survival data were correlated with the staining results. RESULTS: Bcl-2 protein expression (P=0.0472), stage (P=0.0087) and lymph-node involvement (P=0.0488) were found to be independent prognostic factors. Increased bcl-2 protein expression correlated with a better 5-year survival (P=0.0472). Patients who were bcl-2(-)/p53(-) (n=25) or bax(+)/bcl-2(-) (n=13) had a significantly worse overall survival (P=0.0305 and P=0.0482, respectively). Similarly, patients who were bax(+)/bcl-2(-)/p53(-) (n=11) also had a worse 5-year survival compared with the rest of the group (P=0.0088). Changes that were noticed in bax and p53 protein expression from the time of biopsy until the time of surgery did not correlate with a significant increase in the overall survival. CONCLUSIONS: The expression of bcl-2 gene appears to be an independent prognostic factor for patients with laryngeal carcinoma. The coexpression of the genes studied can be used to determine aggressive clinical phenotypes. Copyright 2001 Harcourt Publishers Limited. PMID: 11520092 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Head Neck. 2001 Apr;23(4):280-5. Prognostic significance of Bcl-2 and p53 expression in advanced laryngeal squamous cell carcinoma. Friedman M, Lim JW, Manders E, Schaffner AD, Kirshenbaum GL, Tanyeri HM, Caldarelli DD, Coon JS. Department of Otolaryngology, Rush Medical College of Rush University, Rush-Presbyterian-St. Luke's Medical Center, 30 North Michigan Avenue, Suite 1107, Chicago, Illinois 60602, USA. BACKGROUND: Proteins regulating the cell cycle and cell death are frequently abnormally expressed in cancer. Several of these, particularly p53 and Bcl-2, have been widely suggested as possible prognostic markers in diverse human malignancies. Their role in predicting outcome in squamous cell carcinomas of the head and neck is unclear and may depend on the location, stage, and treatment of the tumor. METHODS: To assess this question specifically for advanced squamous cell carcinoma of the larynx, we studied 69 patients with stage III or IV tumors, all but 6 of whom were treated with surgery plus postoperative irradiation by a single physician. We studied the patients retrospectively to test the association between expression of Bcl-2 and p53, as assessed by immunohistochemistry, with treatment outcome and survival. RESULTS: Twenty of the 69 patients died from their tumor (poor outcome); the rest were alive and tumor free at the last follow-up or died of unrelated causes without clinical tumor recurrence (good outcome). Fourteen tumors had detectable Bcl-2 expression, including 8 scored as overexpressors. Thirty-nine tumors overexpressed p53. Expression of neither Bcl-2 nor p53 was associated with outcome, overall survival, or disease-free survival. Only tumor stage was significantly associated with outcome and disease-free survival. CONCLUSION: These data indicate that assessing expression of p53 or Bcl-2 is unlikely to be prognostically useful for surgically treated advanced laryngeal carcinoma. PMID: 11400228 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Eur J Clin Invest. 2001 Mar;31(3):240-7. Low frequency of p53 and ras mutations in bile of patients with hepato-biliary disease: a prospective study in more than 100 patients. Muller P, Ostwald C, Puschel K, Brinkmann B, Plath F, Kroger J, Barten M, Nizze H, Schareck WD, Hauenstein K, Liebe S, Lohr JM. University of Rostock, Rostock, Germany. The diagnosis of biliary disease, namely malignant disorders, is frequently hampered by the inconclusive cytology. We investigated prospectively the frequency of molecular changes in p53 and ras compared with cytology in patients with primary or secondary hepato-biliary disease. We investigated 118 consecutive patients, aged 24-89 with the following clinical diagnoses: choledocho/cholecystolithiasis (28), cholangiocellular carcinoma (21), gall bladder tumor (8), liver metastasis (3), autoimmune disease (8), chronic pancreatitis (16), pancreatic carcinoma (11), papillary disease (4), hepatic cirrhosis (6), cholangitis (2), anomalies (2), and normal (9). Bile was aspirated during routine endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). DNA was prepared freshly from a native aliquot. p53 mutations were detected by polymerase chain reaction (PCR) for exons 5 through 8 followed by TGGE. PCR for ras mutations was performed as RFLP-PCR with sequencing. In four cases, mutations in p53 could be found in exons 6 and 7. Twenty-two samples showed ras mutations; ras mutations were found in choledocholithiasis (4/28), bile duct (5/21), gall bladder (3/8) and pancreatic (1/11) carcinoma, liver metastasis (3/3), ulcerative colitis (2/3), PSC (1/2), and chronic pancreatitis (1/16). Cytology was clearly positive in seven cases, suspicious in three other, inconclusive in six, and negative in the rest. The molecular analysis resulted in a sensitivity of 33% and specificity of 87%, respectively, for the diagnosis of a malignant condition. PCR for p53 and ras mutations may aid the diagnosis of primary and secondary (metastatic) hepatobiliary disease if a malignant condition of the bile ducts and the liver is suspected and cytology is inconclusive or negative. However, the incidence of p53 and ras mutations in bile seems less frequent than in other malignant conditions of the gastrointestinal tract and the pancreas and lower than in tissue, leaving a poor sensitivity and specificity. Nevertheless, the presence of a p53 and/or ras mutation per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or inconclusive. PMID: 11264652 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Anal Quant Cytol Histol. 2001 Feb;23(1):27-30. Terminal uridine nick end labelling and mitosis in breast carcinoma. Correlation with tumor grade and p53 overexpression. Dey P, Luthra UK, George SS, Haji BI, George J. Cytology Unit, Department of Pathology, Mubarak Al Kabeer Hospital, and Faculty of Medicine, Kuwait University, Kuwait. pranab@ch1.dot.net.in OBJECTIVE: To study the relationship of cell death and proliferation to histologic grade and p53 expression in invasive carcinoma of the breast. STUDY DESIGN: A total of 31 cases of infiltrating duct carcinoma of the breast were randomly selected. The terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) reaction and p53 immunostaining were performed on representative paraffin-embedded tissue sections. Mitotic and apoptotic indices (MI and AI) were also measured on hematoxylin-eosin-stained sections. Histologic grade of infiltrating duct carcinoma was performed with the help of the Nottingham modification of the Bloom-Richardson system. Tumor grade and p53 overexpression were correlated with MI, AI and AI detected by TUNEL. RESULTS: There were a total of 31 infiltrating duct carcinomas of the breast, of which 13 cases were grade 1 and nine cases each were grade 2 and 3. Cells with positive TUNEL showed a strong brown nuclear positivity. TUNEL showed positivity from the periphery of the nuclear margin to the central portion. AI detected by TUNEL did not correlate with tumor grade (ANOVA, P > .05). MI was significant only in grade 1 versus grade 3 and 2 versus grade 3 carcinomas (ANOVA, P < .01). The morphologic apoptotic index was significant only in grade 1 versus grade 3 carcinomas. Nine cases showed p53 overexpression, and the rest of the cases were negative for p53 immunostaining. MI, AI and TUNEL were not significantly different in p53-negative and -positive groups. Pearson's correlation coefficient showed that AI and MI were significantly related, but there was no significant relation between AI detected by TUNEL and MI. CONCLUSION: MI is still more useful than AI or AI detected by TUNEL in differentiating various grades of carcinoma of the breast. PMID: 11233740 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Proteins. 2000 Nov 15;41(3):288-98. Mechanism of protein folding. Nolting B, Andert K. Prussian Private Institute of Technology at Berlin, Berlin, Germany. nolting@pitb.de The high structural resolution of the main transition states for the formation of native structure for the six small proteins of which Phi-values for a large set of mutants have become available, barstar, barnase, chymotrypsin inhibitor 2, Arc repressor, the src SH3 domain, and a tetrameric p53 domain reveals that for the first 5 of these proteins: (1) Residues that belong to regular secondary structure have a significantly larger average fraction of native structural consolidation than residues in loops; (2) on the other hand, secondary and tertiary structures have built up to the same degree, or at least a high degree, but nonuniformly distributed over the molecule; (3) the most consolidated parts of each protein molecule in the transition state cluster together, and these clusters contain a significantly higher percentage of residues that belong to regular secondary structure than the rest of the molecule. These observations further reconcile the framework model with the nucleation-condensation mechanism for folding: The amazing speed of protein folding can be understood as caused by the catalytic effect of the formation of clusters of residues which have particularly high preferences for the early formation of regular secondary structure in the presence of significant amounts of tertiary structure interactions. PMID: 11025541 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Oncogene. 2000 May 18;19(22):2714-20. Proneness to UV-induced apoptosis in human fibroblasts defective in transcription coupled repair is associated with the lack of Mdm2 transactivation. Conforti G, Nardo T, D'Incalci M, Stefanini M. Dipartimento di Oncologia, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea, 62, 20157 Milano, Italy. The apoptotic response and the level of expression of p53 and of three genes transcriptionally activated by p53 (Mdm2, p21 and bax) were investigated in UV-sensitive cells from patients with xeroderma pigmentosum (XP) or Cockayne syndrome (CS). These disorders are due to different genetic defects affecting transcription-coupled repair (TCR) and/or global genome repair (GGR), the nucleotide excision repair subpathways which remove UV-induced lesions from the transcribed strand of active genes or from the rest of the genome, respectively. After 20 J/m2 UV light, normal and GGR-defective XP-C fibroblasts showed rapid increase in p53, late induction of Mdm2 and no evidence of apoptosis even 96 h after irradiation. In contrast, in XP-A (defective in GGR and TCR), CS-A and CS-B (defective only in TCR) fibroblasts, the p53 increase was not followed by Mdm2 induction and the persistence of high levels of p53, due to the lack of its degradation by Mdm2, was associated with the appearance of apoptosis. Besides indicating that the persistence of DNA damage in the transcribed strand of active genes leads to apoptosis, these findings provide the first evidence that the lack of activation of Mdm2 plays a key role in the cascade of events leading to apoptosis. Oncogene (2000). PMID: 10851071 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Hepatology. 2000 Apr;31(4):885-9. Detection of adenovirus and initiation of apoptosis in hepatocellular carcinoma cells after Ad-p53 treatment. Mitry RR, Sarraf CE, Havlik R, Habib NA. Liver Surgery Section, Imperial College School of Medicine, Hammersmith Hospital Campus, London, UK. Transcription of the p53 gene can regulate progression of apoptosis in a wide variety of tissues. Three categories of human hepatocyte culture have been used to show the initiation of apoptosis after treatment with p53-bearing adenovirus. Chang liver cells are derived from normal liver tissue and express native p53, whereas hepatocellular carcinoma (HCC)-derived cell lines were Hep3B (p53-deleted) and PLC/PRF/5 (p53-mutant). Cultures were infected with Ad-p53 (15 particles per cell; 36 hours), and after treatment, morphological changes in all cell categories were observed by electron microscopy. Infection was evident in the cytoplasm of all treated cell types: after entry across the plasma membrane viruses translocated and came to rest surrounding and adjacent to nuclei, cytoplasm proximal to nuclear membranes became dense with virus- and membrane-derived debris, but intact viruses did not enter nuclei. Apoptosis, recognized morphologically by characteristic chromatin and cytoplasmic condensation, occurred more frequently in HCC-derived cells, and the ultimate fate of apoptotic bodies was phagocytosis and degradation by neighboring cells. PMID: 10733544 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Ann N Y Acad Sci. 1998 Nov 20;854:318-27. Telomeres: influencing the rate of aging. von Zglinicki T. Institute of Pathology, Charite, Humboldt University, Berlin, Germany. zglinick@rz.charite.hu-berlin.de Evidence is reviewed that suggests a central role for telomeres in one major model of biological aging, namely, proliferative senescence. Telomeric shortening with each cell division does not only act as a biological clock, but appears to trigger the ultimate loss of proliferative ability via activation of the p53-dependent check point system. Oxidative stress induces single-stranded damage in telomeric DNA. It is not clear yet whether this damage occurs in the form of single-stranded gaps or overhangs or as arbitrarily distributed single-stranded breaks. However, in contradiction to the rest of the genome, this damage is not repaired in telomeres. It is, therefore, the major cause of telomere shortening even under standard in vitro cell culture conditions. Therefore, controlling the oxidative load onto DNA, in general, and, especially, onto telomeres might become a major factor to influence the rate of aging. Further experiments demonstrate that G-rich single-stranded telomeric DNA fragments do activate the p53 check point control, leading to an inhibition of proliferation in wild-type p53 cells. Not only the shortening of telomeres down to a "signal value," but accumulation of telomeric single-stranded DNA fragments, as well, could be relevant triggers for proliferative senescence. Publication Types: Review Review, Tutorial PMID: 9928440 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Brain Res. 1999 Feb 6;818(1):23-33. Apoptosis and expression of p53 response proteins and cyclin D1 after cortical impact in rat brain. Kaya SS, Mahmood A, Li Y, Yavuz E, Goksel M, Chopp M. Department of Neurosurgery, Henry Ford Health Sciences Center, Detroit, MI, USA. We measured the temporal profile and cellular identification of apoptosis in rat brain after cortical contusion injury. Double staining immunohistochemistry was also used to investigate the relationship between apoptotic cell death and selective protein expression associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd45, PCNA) and cell cycle protein, Cyclin D1, in male Wistar rats 48 h after injury. Cortical contusion was induced in male Wistar rats with a pneumatic impactor device. The animals were sacrificed at different times after trauma (1, 2, and 14 h and 1, 2, 4, 7 and 14 days; n=4 per time point). Sham-operated rats (n=4) and normal rats not subjected to any surgical procedure (n=4) were used as controls for temporal profile determination. Additional 11 rats were used for study of protein expression. Coronal brain sections were analyzed using an in situ terminal deoxynucleotdyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL), hematoxylin, and immunohistochemical double staining methods. Apoptotic cells were observed as early as 2 h after the impact. Apoptotic cell death peaked at 2 days, gradually tapering off afterward, although scattered apoptotic cells were detected at 2 weeks after the impact. The number of apoptotic cells at 2 days far exceeded their number at other times (p=0.009). Apoptotic cells were observed primarily in the cortex adjacent to the site of injury. In addition, apoptotic cells in conjunction with few injured cells were present in the ipsilateral hippocampus and localized to the granule layer of dentate gyrus. Our data indicate that DNA fragmentation is present in nearly all neurons subacutely after cortical contusion and persists for at least 2 weeks thereafter. Apoptosis is also present in neurons localized to the hilus of the dentate gyrus at a site remote from the area of injury suggesting a selective role for apoptosis in promoting secondary brain damage and dysfunction after traumatic brain injury. Using double staining, we were able to show that a great majority of apoptotic cells (>95%) were neurons and the rest were astrocytes and endothelial cells. Proteins associated with DNA damage and repair (p53, Bax, MDM2, WAF1, Gadd 45, PCNA) were expressed in the cytoplasm of normal cells of naive and sham rats. These proteins were translocated to the nuclei of apoptotic and injured cells at 48 h after cortical contusion. Cyclin D1 was not present in apoptotic cells. The differential expression of proteins associated with DNA damage, repair and the cell cycle protein Cyclin D1 in the contused brain suggest a potential role for these proteins in cell survival and apoptosis after cortical contusion. Copyright 1999 Elsevier Science B.V. PMID: 9914434 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):14675-80. Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. Nikolova PV, Henckel J, Lane DP, Fersht AR. Cambridge University Chemical Laboratory and Cambridge Centre for Protein Engineering, Medical Research Council Centre, Cambridge CB2 2QH, United Kingdom. We have designed a p53 DNA binding domain that has virtually the same binding affinity for the gadd45 promoter as does wild-type protein but is considerably more stable. The design strategy was based on molecular evolution of the protein domain. Naturally occurring amino acid substitutions were identified by comparing the sequences of p53 homologues from 23 species, introducing them into wild-type human p53, and measuring the changes in stability. The most stable substitutions were combined in a multiple mutant. The advantage of this strategy is that, by substituting with naturally occurring residues, the function is likely to be unimpaired. All point mutants bind the consensus DNA sequence. The changes in stability ranged from +1.27 (less stable Q165K) to -1.49 (more stable N239Y) kcal mol-1, respectively. The changes in free energy of unfolding on mutation are additive. Of interest, the two most stable mutants (N239Y and N268D) have been known to act as suppressors and restored the activity of two of the most common tumorigenic mutants. Of the 20 single mutants, 10 are cancer-associated, though their frequency of occurrence is extremely low: A129D, Q165K, Q167E, and D148E are less stable and M133L, V203A and N239Y are more stable whereas the rest are neutral. The quadruple mutant (M133LV203AN239YN268D), which is stabilized by 2.65 kcal mol-1 and Tm raised by 5.6 degreesC is of potential interest for trials in vivo. PMID: 9843948 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: Radiother Oncol. 1998 May;47(2):175-83. Nodal CT density and total tumor volume as prognostic factors after radiation therapy of stage III/IV head and neck cancer. Grabenbauer GG, Steininger H, Meyer M, Fietkau R, Brunner T, Heinkelmann P, Hornung J, Iro H, Spitzer W, Kirchner T, Sauer R, Distel L. Department of Radiation Therapy, University Hospitals of Erlangen, Germany. PURPOSE: To determine whether the immunohistochemical expression of proliferation-associated antigens (proliferating cell nuclear antigen, MIB1) and the nuclear p53 reactivity in addition to total tumor volume, nodal CT density and T and N category are predictive for overall survival and locoregional tumor control in patients with squamous cell carcinoma of the head and neck region. MATERIALS AND METHODS: Between October 1989 and September 1993, 87 patients with biopsy proven head and neck cancer were randomly allocated to receive radiation alone or simultaneous radiation and chemotherapy as part of a multicenter trial with a total of 298 randomized patients. There were only inoperable lesions in UICC (1992) stage III (8%) and IV (92%). Radiotherapy was delivered with 180 cGy twice daily up to a total dose of 7020 cGy in 51 days. Three cycles of 2340 cGy each were separated by a rest period of 11 days. Chemotherapy consisted of cis-DDP, 5-fluorouracil and leucovorin and was repeated on days 22 and 44. Routinely-processed paraffin-embedded sections were stained using monoclonal antibodies for detection of proliferation-associated antigens (MIB1 and PCNA) and p53 oncoprotein to determine the labeling index (LI). In addition, the total tumor volume and the percentage of necrosis were measured using CT data. The median follow-up was 3.9 years (range 1.9-5.0 years). RESULTS: The overall survival and locoregional control for all 87 patients were 34 and 39% at 3 years, respectively. The addition of chemotherapy resulted in a better overall survival (27 versus 47%, P = 0.03) but did not influence locoregional control (31 versus 47%, P = 0.08). In univariate analysis, nodal CT density (P < 0.0001), total tumor volume (P < 0.0001), age (P = 0.001) and the MIB1-LI (P = 0.04) had a significant impact on overall survival. However, in the final Cox model only the nodal CT density (P = 0.0003) and age (P = 0.05) were independent prognostic factors for survival and only the nodal CT density (P = 0.0006) was an independent prognostic factor for locoregional control. The expression of the p53 oncoprotein was not found to have a clear predictive value. CONCLUSION: Nodal CT density, total tumor volume and age will remain the relevant prognostic factors in stage III/IV head and neck cancer. Publication Types: Clinical Trial Multicenter Study Randomized Controlled Trial PMID: 9683366 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: Cell Struct Funct. 1998 Apr;23(2):101-7. Cell lines established from fetal brains of p53-deficient mice. Tomooka Y, Aizawa S. Department of Biological Science and Technology, Science University of Tokyo, Chiba, Japan. tomoylab@rs.noda.sut.ac.jp Brains from 7 p53-deficient mice on fetal day 13 were divided into 4 regions: cerebral cortex, cerebellum, upper spinal cord, and the rest of brain. They were trypsinized and cultured in medium containing 10% fetal calf serum. By dilution culture, 138 clonal lines were established from every regions. The lines were characterized morphologically and immunocytochemically as neuronal, glial, myogenic, or unidentified. They were cultured for more than a year, indicating that the lines are immortalized. p53-deficiency alone is sufficient for establishing clonal cell lines of the central nervous system. PMID: 9669038 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Clin Cancer Res. 1998 May;4(5):1345-55. Higher potency of N-(4-hydroxyphenyl)retinamide than all-trans-retinoic acid in induction of apoptosis in non-small cell lung cancer cell lines. Zou CP, Kurie JM, Lotan D, Zou CC, Hong WK, Lotan R. Department of Tumor Biology, The University of Texas M. D. Anderson Cancer Center, Houston 77030, USA. Most human non-small cell lung cancer (NSCLC) cell lines are refractory to all-trans-retinoic acid (ATRA). Recently, N-(4-hydroxyphenyl)retinamide (4HPR) was found to induce apoptosis in various tumor cells. In this study, we compared and contrasted the effects of 4HPR and ATRA on the growth and apoptosis of 10 NSCLC cell lines and normal human bronchial epithelial (NHBE) cells. All of the cancer cell lines and the NHBE cells were sensitive to 10 microM 4HPR, and their numbers decreased to <20% of the controls after a 5-day treatment, whereas ATRA decreased cell numbers to about 50% of the controls in three cell lines and was less effective in the rest of the tumor cell lines. ATRA inhibited the growth of the NHBE cells by 70-80%. 4HPR induced apoptosis in most of the cells, including the ATRA-resistant ones, as evidenced by a DNA fragmentation assay. No correlation was found between growth inhibition by 4HPR and the expression of retinoic acid receptor beta (determined by Northern blotting and PCR), p53, or Bcl-2 proteins (analyzed by Western blotting). These results demonstrate that 4HPR is more potent than ATRA in inducing apoptosis in NSCLC cells and suggest that further clinical trials for prevention and therapy of NSCLC using 4HPR are warranted. PMID: 9607596 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Jpn J Cancer Res. 1998 Mar;89(3):269-77. p53 gene mutation and loss of heterozygosity of chromosome 11 in methylcholanthrene-induced mouse sarcomas. Shimokado K, Watanabe H, Sumii M, Miyagawa K, Kamiya K, Dohi K, Niwa O. Second Department of Surgery, School of Medicine, Hiroshima University. Mutations of the p53 tumor suppressor gene are the most prevalent genetic alteration observed in a wide variety of human cancers. In this study we examined 63 methylcholanthrene (MCA)-induced sarcomas from C57BL/6N x C3H/HeN F1 (BCF1) or C3H/HeN x C57BL/6N F1 (CBF1) mice for p53 gene mutations and loss of heterozygosity (LOH) of chromosome 11. Mutation analysis was done on exons 5 to 8 of the p53 gene by polymerase chain reaction-single strand conformation polymorphism analysis. This identified 53 potential mutations in 45 sarcomas. Mutations were further confirmed by direct sequencing of the region. Forty-nine of the 53 cases (94%) were missense mutations, while the rest included two nonsense mutations, one silent mutation and one insertional mutation. Spectra of base substitutions were: 25 cases (47%) of G:C-->T:A transversion, 13 cases (25%) of G:C-->A:T transition (CpG site 15%), 13 cases (24%) of G:C-->C:G transversion, a case (2%) of A:T-->T:A transversion and a case (2%) of insertion. In addition, analysis of 5 polymorphic markers of mouse chromosome 11 revealed LOH in ten cases (22%) among those carrying p53 mutations. In nine of these 10 cases, the loss involved all 5 markers. In addition, the loss was biased toward the C57BL allele (9 cases). The present study establishes the pattern of mutation of the p53 gene in MCA-induced mouse sarcomas. PMID: 9600120 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: J Clin Endocrinol Metab. 1998 May;83(5):1801-5. Comparative genomic hybridization analysis of nonfunctioning pituitary tumors. Daniely M, Aviram A, Adams EF, Buchfelder M, Barkai G, Fahlbusch R, Goldman B, Friedman E. Institute of Genetics, Chaim Sheba Medical Center, Tel-Hashomer, Israel. Clinically nonfunctioning pituitary adenomas constitute about one third of pituitary neoplasms and are considered monoclonal tumors. The molecular mechanisms of tumorigenesis in these neoplasms are poorly understood, as evidenced by the paucity of reported somatic genetic alterations. Furthermore, the somatic mutations detected to date were primarily ascribed to candidate genes or chromosomal regions: gsp, ras, p53 mutations, and allelic losses of 11q and 13q. To gain insight into which chromosomal regions bear genes involved in nonfunctioning pituitary tumorigenesis, we examined 23 such tumors by comparative genomic hybridization. Four tumors showed no genetic abnormality, and the rest (17 of 23, 74%) exhibited at least one chromosomal region of abnormality. Gains and losses affected all chromosomes (except for chromosome 14). Notably, 8 of 23 tumors (34.7%) displayed sex chromosome and chromosome 18 aberrations (amplifications or deletions). Nonrandom DNA amplification of sub-chromosomal regions on 4q, 5q (5q13-->5q23), 9p (9p21-->9pter), 13q (13q21-->13q32), and 17q were detected in 10-30% of the tumors. Noteworthy, no tumor displayed deletion of 11q, the MEN1 gene locus. These findings suggest that genes localized to previously undescribed chromosomal regions play a role in the tumorigenesis of nonfunctioning pituitary adenomas. PMID: 9589696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Mol Biol. 1998 Jan 30;275(4):575-88. The N terminus of the murine p53 tumour suppressor is an independent regulatory domain affecting activation and thermostability. Hansen S, Lane DP, Midgley CA. Department of Biochemistry, University of Dundee, Scotland, UK. The contribution of each of the structural domains of p53 to its function has been discussed widely in the literature. Crystallographic studies have revealed much about the structure of the core DNA binding domain, but as it has not been possible to use this approach for the intact protein, the effect of the domains flanking the core must be investigated by more indirect techniques. In this study a series of truncated murine p53 proteins has been investigated for DNA binding activity at 4 degrees C and 37 degrees C, transcriptional activation, and tumour suppression activity. Full-length p53, and truncations lacking the N terminus, purified from a baculovirus expression system all show latency for DNA binding; that is, they must be activated to bind by association with a C-terminal antibody such as PAb421. This demonstrates that latency for DNA binding is independent of the N terminus. Truncations lacking the C-terminal oligomerisation domain, and the isolated core domain, can only be activated to bind DNA and PAb1620 (an antibody recognising the wild-type conformation of the core domain) in the presence of cross-linking antibodies, while murine core only binds to DNA in the presence of PAb1620. An analysis of the thermostability of DNA binding revealed that antibodies that bind the N terminus of p53 could protect the protein against loss of activity at 37 degrees C. C-terminal antibodies, however, were ineffective unless the N-terminal 37 amino acid residues were absent. The N terminus may retain some secondary structure, since it is the main contributor to the anomalous migration in SDS-polyacrylamide gels. Our results suggest that the N terminus has a destabilising effect that influences conformation of p53 at 37 degrees C, so cellular proteins binding to the N terminus in vivo may modulate p53 conformation and stability. The effects on thermostability are also direct evidence showing that antibodies binding to N-terminal deletions create a conformational change in the rest of the molecule. In addition, longer deletions of the C terminus reduce the ability of p53 to transactivate target genes and inactivate tumour suppression activity, while truncations of the N terminus retain partial tumour suppression activity. Our results clearly show participation of both the N and C termini in the regulation of all the functions of p53 at 37 degrees C, indicating that distinct, independent domains interact with each other within, the flexible structure of p53. PMID: 9466932 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 31: Biochem Biophys Res Commun. 1998 Jan 14;242(2):317-21. Cyclin E overexpression responsible for growth of human hepatic tumors with p21WAF1/CIP1/SDI1. Tsuji T, Miyazaki M, Fushimi K, Mihara K, Inoue Y, Ohashi R, Ohtsubo M, Hamazaki K, Furusako S, Namba M. Department of Cell Biology, Institute of Molecular and Cellular Biology, Okayama University Medical School. We examined a relationship between p21WAF1/CIP1/SDI1 and cell-cycle-related proteins in 12 human liver tumor cell lines (JHH-1, -2, -4, -5, -6, -7; HLE; HuH-7; Hep3B; PLC/PRF/5; HuH-6; HepG2). Seven (JHH-1, -2, -5, -6, -7; Hep3B; HepG2) out of eight cell lines having p21WAF1/CIP1/SDI1 protein overexpressed cyclin E protein, although one of them (JHH-5) overexpressed a reduced size of cyclin E. The rest (HuH-6) of the 8 cell lines with p21WAF1/CIP1/SDI1 showed a decreased expression of cyclin E. Four cell lines (JHH-4; HLE; HuH-7; PLC/PRF/5) deficient of p21WAF1/CIP1/SDI1 protein did not overexpress cyclin E protein. As to expression of the other cell-cycle-related proteins, cyclin A, cyclin D1, CDK2 or CDK4, no significant difference was detected among the 12 cell lines. These findings indicate that the human liver tumor cell lines which have the p21WAF1/CIP1/SDI1-inducible barriers of the cell cycle progression can go through the G1/S checkpoint by overexpressing cyclin E. PMID: 9446792 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 32: Am J Physiol. 1997 Sep;273(3 Pt 1):L572-80. Cell cycle in alveolar epithelial type II cells: integration of Matrigel and KGF. Buckley S, Driscoll B, Anderson KD, Warburton D. Childrens Hospital Los Angeles Research Institute, California 90027, USA. The regulation of cell cycle control in alveolar epithelial type II cells (AEC2) in response to peptide growth factors and extracellular matrix signals is not well understood. Herein, we have determined that, in adult rat AEC2 in primary culture on Engelbreth-Holm-Swarm biomatrix (Matrigel) in the presence of keratinocyte growth factor, the expression of key cell cycle control elements, including cyclins A and D and cyclin-dependent kinases (cdk) 1 and 4, is increased and that retinoblastoma protein (pRb) phosphorylation is also increased, with a corresponding decrease in the expression of p53 and the cdk inhibitors (cdkis) p21WAF1/CIP1 and p27KIP-1 compared with cells cultured on plastic. The Matrigel biomatrix-KGF culture conditions were also associated with an enhanced proliferative response, as measured by fluorescent-activated cell sorter analysis, thymidine incorporation into DNA, and proliferating cell nuclear antigen expression. This enhanced proliferation occurred with neither a soluble extract of Matrigel biomatrix nor with other simple biological matrices. We conclude that coordinated induction of key cyclins and cdks, with the concomitant suppression of key negative cell cycle regulators, occurs in AEC2 on Matrigel biomatrix in the presence of KGF. We speculate that the balance between cyclin and cdk activation and cdki suppression in AEC2 serves to integrate the combined influences of biomatrix and KGF signaling on pRb phosphorylation, thereby controlling transit through S phase of the cell cycle. Conversely, AEC2 express high levels of cdkis and p53 at rest in G1 phase. The latter finding may explain the quiescent state of normal adult AEC2 in vivo. PMID: 9316491 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 33: Exp Cell Res. 1997 Aug 25;235(1):287-94. Effect of the insulin-like growth factor I receptor on ionizing radiation-induced cell death in mouse embryo fibroblasts. Nakamura S, Watanabe H, Miura M, Sasaki T. Faculty of Dentistry, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo, 113, Japan. We have investigated the effect of the insulin-like growth factor I receptor (IGF-IR) on ionizing radiation (IR)-induced cell death using the following two mouse embryo fibroblast cell lines: (i) R- cells with a null mutation of the IGF-IR gene, therefore expressing no endogenous IGF-IR; (ii) R+ cells derived from R- cells, a stable transfectant overexpressing the human IGF-IR. Numbers of R- cells began to detach from dishes and float into the medium about 48 h after 10 Gy of X-irradiation. Internucleosomal DNA fragmentation detected by agarose gel electrophoresis, which is characteristic of apoptosis, was observed in the floating R- cells, but not in the attached cells. Unexpectedly, morphological analysis of the floating cells 72 h after irradiation revealed that only about half of them showed apoptotic death and the rest showed a nonapoptotic, presumably necrotic, one. On the other hand, R+ cells retained more than 90% viability even 4 days after irradiation, and very few floating cells were observed. The G2 arrest was induced in both cell lines following irradiation and G2/M fractions similarly returned to normal levels by around 20 h after irradiation, indicating that the cell death which appeared thereafter in R- cells is mediated through mitosis. Significant induction of p53 following irradiation was not detected by Western blot analysis in either R- or R+ cells. Collectively, these results demonstrate that signal transduction pathways originating from the IGF-IR may be involved in preventing IR-induced apoptosis and necrosis without affecting cell cycle arrest or p53 pathways. PMID: 9281378 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 34: Arterioscler Thromb Vasc Biol. 1997 May;17(5):947-53. Cellular radiosensitivity, radioresistant DNA synthesis, and defect in radioinduction of p53 in fibroblasts from atherosclerosis patients. Nasrin N, Mimish LA, Manogaran PS, Kunhi M, Sigut D, Al-Sedairy S, Hannan MA. Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Earlier studies have suggested that both cancer and atherosclerosis may follow a common pathway in the early stage of development and share certain risk factors. One report indicated that the gene responsible for the radiosensitive, cancer-prone, multisystem disorder ataxia telangiectasia (AT) may increase the risk of developing ischemic heart disease. The present studies were carried out to find similarities, if any, between atherosclerosis patients and AT homozygotes or heterozygotes (ATHs) in their cellular/molecular response to ionizing radiation, which acts as a carcinogen as well as an atherogen. Fibroblast cell strains developed from healthy subjects and from AT homozygotes, ATHs, and atherosclerosis patients were compared for (1) survival, by the colony-forming assay and (2) DNA synthesis inhibition after irradiation, determined by [3H]thymidine incorporation, cell cycle distribution, and the expression of p53 and p21 proteins, analyzed by flow cytometry. Fibroblasts from the atherosclerosis patients as a group, compared with the healthy subjects, showed enhanced sensitivity to chronic (low-dose-rate) irradiation. A majority of the cell strains representing atherosclerosis patients exhibited varying degrees of radioresistant DNA synthesis (RDS), with roughly 33% showing an AT-like and the rest an ATH-like response. All cell strains with an AT-like and one quarter with an ATH-like RDS were found to be defective in the radioinduction of both p53 and p21 proteins, which are concerned with cell cycle regulation. An absence of G1 arrest after irradiation was observed in cell strains lacking a radioinduced expression of p53 and p21. Cellular/molecular defects leading to increased radiosensitivity, reduced induction of p53/p21, and cell cycle deregulation found to be associated with cancer-prone disorders such as AT may constitute important risk factors for atherosclerosis as well. PMID: 9157960 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 35: Ann Med. 1997 Apr;29(2):121-6. The role of apoptosis in gynaecological malignancies. Sheets EE, Yeh J. Department of Obstetrics and Gynecology, Brigham and Women's Hospital, Boston, MA 02115, USA. eesheets@bics.bwh.harvard.edu Apoptosis is a process of single-cell deletion requiring active participation of the cell in its own demise. First described in 1972, it is now known to play a major role in embryogenesis, tissue homeostasis and neoplasia. Apoptosis can be initiated when DNA damage occurs causing the cell to pause in its reproductive cycle. If the DNA damage is beyond repair, the cell proceeds to apoptotic cell death. When the genetic mechanism(s) involved in the pathway of apoptosis is altered, the cell does not die. Further mutations occur by proliferation and such multiple mutational events can lead to a malignant phenotype and cancer growth. The tumour suppressor gene p53 causes a DNA-damaged cell to rest and attempt repair. If damage is irreparable, p53 levels will continue to increase, initiating apoptosis. Mutation of p53, found in approximately 50% of cancers, can stop the apoptotic process. Increased bcl-2 expression, an apoptosis inhibitor, also plays a role in cellular transformation and cancer growth. Its altered expression occurs in the presence of oncogene expression. This paper reviews the role of apoptosis in malignant transformation, cancer growth, and response to therapy for gynaecological cancers. For cervical cancer and its precursors, data on apoptotic index, bcl-2 and Bax expression are presented and discussed in relationship to human papillomavirus expression. In ovarian epithelial malignancies, the role that apoptosis plays in chemotherapeutic responses is reviewed. The data for endometrial cancer are currently limited to apoptotic index. Publication Types: Review Review, Tutorial PMID: 9187226 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 36: Cancer Genet Cytogenet. 1996 Sep;90(2):118-24. Trisomy 12 and p53 deletion in chronic lymphocytic leukemia detected by fluorescence in situ hybridization: association with morphology and resistance to conventional chemotherapy. Cano I, Martinez J, Quevedo E, Pinilla J, Martin-Recio A, Rodriguez A, Castaneda A, Lopez R, Perez-Pino T, Hernandez-Navarro F. Department of Hematology and Hemotherapy, Hospital La Paz, Madrid, Spain. The incidence of trisomy 12 and p53 deletion was studied in a group of chronic B-lymphocytic leukemia (B-CLL) patients, using fluorescence in situ hybridization (FISH). Trisomy 12 was detected in eight of 50 patients (16%) and p53 deletion in six of 38 cases analyzed (15.8%). A statistically significant difference was observed between the incidence of trisomy 12 in patients with typical and atypical morphology (3.03% versus 41.18%). No correlation was found between this alteration and the rest of the clinical and biological parameters studied (adenopathies, hepatomegaly, splenomegaly, lymphocyte count, staging, CD11c expression, and resistance to chemotherapy). The p53 deletion was correlated with the presence of hepatomegaly and splenomegaly, advanced stage of disease, and resistance to conventional chemotherapy. The application of FISH to whole blood cell nuclei, without prior manipulation or culture, showed a higher percentage of cells with trisomy 12 than when the method was used following culture. We conclude that 1) FISH is a simple and sensitive technique for the detection of numerical and structural chromosome abnormalities; 2) Its application to uncultured samples obviates the alteration of results originated by the probable growth advantage of the normal or neoplastic cell population in vitro; 3) Trisomy 12 appears to define a B-CLL subgroup of atypical morphology; and 4) The p53 deletion is correlated with advanced stage of disease and resistance to treatment. PMID: 8830719 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 37: J Surg Oncol. 1996 Jun;62(2):86-92. Node negative breast carcinoma: hyperprolactinemia and/or overexpression of p53 as an independent predictor of poor prognosis compared to newer and established prognosticators. Patel DD, Bhatavdekar JM, Chikhlikar PR, Ghosh N, Suthar TP, Shah NG, Mehta RH, Balar DB. Division of Research, Gujarat Cancer Society, Ahmedabad, India. The purpose of this study was to investigate a prognostic indicator that can differentiate node negative breast cancer patients (N = 39, T2N0M0) with high risk and low risk for the development of recurrence or metastases. Preoperative plasma prolactin (PRL) was estimated by radioimmunoassay. The expression of PRL, p53, nm23, and c-erbB2 was investigated by immunohistochemical (IHC) localization; cathepsin D (CD, Enzyme Linked Sorbant Assay) and estrogen- and progesterone-receptors (ER and PR, Dextran coated charcoal method) were estimated in the tumor cytosols. The follow-up period was 2-6 years. Statistical comparisons were made between each marker for relapse-free survival (RFS) and overall survival (OS). Of the 39 patients, 18 had hyperprolactinemia (PRL > 20.0 ng/ml plasma), whereas overexpression of p53 was observed in 55% (17/31) tumors. These were independently and in combination associated with a reduced RFS and OS. The rest of the investigated markers did not show promising results. Hyperprolactinemia and/or overexpression of p53 were associated with aggressiveness of the tumor, early disease relapse or metastases, and poor OS in patients with node negative breast cancer. These two markers may enhance our ability to identify node negative breast cancer patients with aggressive tumors, for whom the use of adjuvant chemo and/or endocrine therapy is unequivocally justified. PMID: 8649046 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 38: Carcinogenesis. 1995 Nov;16(11):2879-81. p53 mutations in primary hepatic angiosarcomas not associated with vinyl chloride exposure. Soini Y, Welsh JA, Ishak KG, Bennett WP. Department of Pathology, University of Oulu, Finland. Angiosarcomas of the liver are rare, malignant cancers composed of neoplastic blood vessels. Human hapatic angiosarcomas have been associated with liver cirrhosis or exposure to vinyl chloride, Thorotrast or arsenic. A recent analysis of six hepatic angiosarcomas associated with vinal chloride exposure found three mutations and all were A:T --> T:A transversions, which are otherwise uncommon in human cancers. To test the specificity of this mutation spectrum, we analyzed 21 hepatic angiosarcomas not associated with vinyl chloride exposure. Four cases were exposed to Thorotrast, none had a history of arsenic exposure and the rest were sporadic. Exons 5-8 of the p53 gene were amplified by polymerase chain reaction, and the products were sequenced directly. Two G:C --> A:T transitions were found in two tumors: TGCstop in codon 136. Neither mutation was associated with Thorotrast exposure. These data indicate that p53 mutations are uncommon in sporadic hepatic angiosarcomas (2/21, 9%), and the mutational profile is consistent with endogenous mechanisms. Both features support the evidence linking vinyl chloride exposure to hepatic angiosarcomas containing an increased frequency of p53 mutations with a mutational spectrum (i.e. A:T --> T:A transversions) characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride. PMID: 7586214 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Cancer. 1995 Oct 1;76(7):1201-8. p53 protein detected by immunohistochemistry as a prognostic factor in patients with epithelial ovarian carcinoma. Klemi PJ, Pylkkanen L, Kiilholma P, Kurvinen K, Joensuu H. Department of Pathology, University Central Hospital of Turku, Finland. BACKGROUND. The clinical significance of p53 suppressor gene nucleoprotein immunostaining in ovarian epithelial cancer has not been determined. METHODS. p53 protein expression was studied by immunohistochemistry from paraffin embedded tissue in a series of 136 patients with malignant ovarian epithelial tumors. The median follow-up time of the patients still alive was 10 years. RESULTS. Sixty (44%) carcinomas stained clearly positive for p53 protein. Positive staining for p53 protein was associated with the serous histologic type (P = 0.0006), a higher than the median S-phase fraction size determined by DNA flow cytometry (P = 0.02), and poor histologic grade of differentiation (P = 0.04), but not with the International Federation of Gynecology and Obstetrics (FIGO) stage, age at diagnosis, or DNA ploidy. Cancers with positive staining had only 17% 5-year and 9% 15-year survival rates compared with 42% 5-year and 36% 15-year survival rates corrected for intercurrent deaths among the rest of patients (P = 0.002). In a multivariate analysis, positive p53 staining was associated with poor survival (relative risk of death, 1.8, 95% confidence interval [CI], 1.2-2.9) together with less than radical surgery (nonradical vs. radical: RR, 5.5; 95% CI, 2.2-13.6), and advanced FIGO stage (RR, 1.4; 95% CI, 1.0-2.0). CONCLUSION. Although p53 protein immunostaining is associated with several other prognostic factors in epithelial ovarian cancer, it may also have independent prognostic value in this disease. PMID: 8630898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Br J Cancer. 1994 Mar;69(3):586-91. p53 protein in low-grade astrocytomas: a study with long-term follow-up. Iuzzolino P, Ghimenton C, Nicolato A, Giorgiutti F, Fina P, Doglioni C, Barbareschi M. Department of Histopathology, Ospedale Civile Maggiore, Verona, Italy. The immunohistochemical expression of p53 protein (p53) was examined in 52 patients out of a series of 66 patients with low-grade astrocytomas with long-term follow-up. All patients were also evaluated for several clinical and histological features, among which only preoperative Karnofsky score and the extent of surgery were statistically significant parameters to predict outcome on multivariate analysis. p53 accumulation was seen in 46.1% of patients, with a wide range of percentage of positive cells. Median survival for p53-positive and p53-negative patients was 41 and 37 months respectively. The survival curves of p53-positive and -negative patients were not statistically different. However, the curves showed a trend towards a more aggressive course in p53-positive patients beginning 3-4 years after surgery. Five years after diagnosis the survival estimate with the Kaplan-Meier method was 21.2% for patients with p53-positive tumours and 45.9% for patients with p53-negative tumours. This trend is not due to different distribution of major clinical prognostic factors (age, incomplete resection or Karnofsky status). The trend could be related to the time needed by the p53-positive clone to outgrow the rest of the p53-negative neoplastic cell population. This hypothesis is further supported by the fact that the five recurrences which were surgically removed (one anaplastic astrocytoma and four glioblastomas) derived from p53-positive tumours and were themselves intensely p53 positive. PMID: 8123492 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Oncogene. 1992 Jul;7(7):1371-81. Correlation between the conformational phenotype of p53 and its subcellular location. Zerrahn J, Deppert W, Weidemann D, Patschinsky T, Richards F, Milner J. Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie Universitat Hamburg, Germany. In order to obtain insight into the parameters determining the subcellular localization of mutant and wild-type forms of p53, we analysed the subcellular distribution of p53 in four Balb/c mouse-derived cell lines ranging in their cellular phenotypes from normal (3T3), via minimal transformant (T3T3), to maximally transformed (3T3tx, Meth A). Epitope mapping showed the p53 proteins in 3T3 and in T3T3 cells to be in a wild-type conformation, as they reacted with PAb246, whereas p53 in 3T3tx and in Meth A cells were PAb246 negative and thus displayed a mutant conformation. Despite its reactivity with PAb246, p53 in T3T3 cells had an extended half-life and accumulated to abnormally high levels. We show that the conformationally wild-type p53 in 3T3 and T3T3 cells predominantly localized to the cell nucleus, with about half of it being tightly associated with nuclear structures. In contrast, approximately 60% of mutant p53 in 3T3tx and Meth A cells localized to the cytoplasm, the rest residing in the cell nucleus; all the nuclear p53 in these cells appeared to be structurally bound. The cytoplasmic location of mutant p53 in 3T3tx and Meth A cells was not seen by immunofluorescence microscopic analysis, and required cell fractionation for its detection. Both cytoplasmic and nuclear p53 of the mutant phenotype bound to hsc proteins with a similar stoichiometry, suggesting that hsc binding is not directly related to the subcellular distribution of these proteins. We suggest that the conformational phenotype of p53 is a major determinant of its subcellular location. PMID: 1620550 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Exp Cell Res. 1988 Dec;179(2):565-74. Early events in murine erythroleukemia cells induced to differentiate: variation of the cell cycle parameters in relation to p53 accumulation. Khochbin S, Chabanas A, Lawrence JJ. Unite INSERM 309, Departement de Recherche Fondamentale, CEN-Grenoble, France. Among the early events of induced differentiation of murine erythroleukemia cells that we studied was the variations of cell distribution in the cell cycle as a function of the time of induction. Flow-cytofluorimetry measurements of DNA content and BrdU incorporation allowed for a precise determination of the variations of the cell cycle parameters. Cells underwent a transient arrest in both G1 and G2 + M between 6 to 16 h of induction. The progression of the cells through S phase seems not to be affected during this period. After this time cells escaped from G1 and reentered the S phase. We described previously [S. Khochbin et al. (1988) J. Mol. Biol. 200, 55-64], that p53 decreased continuously during the induction of MELC and remained at a steady-state level after 18 to 20 h of induction. In order to look for a possible redistribution of the protein along the cell cycle during the induction process, we measured the accumulation of the protein along the cell cycle. In noninduced cells there were four steps in the accumulation of the protein throughout the cell cycle: the amount of p53 was constant during G1 and it increased as cells progressed through S phase, which is characterized by an increased accumulation at the G1/S transition and a more moderate accumulation during progression through the rest of the S phase. A constant level in G2/M, approximately twice that obtained in G1, was achieved. There was no change in this distribution that correlated with the various modifications of the cell cycle in induced cells. It seems then, that p53 is associated neither with the progression of the cells in the S phase nor with the resumption of the DNA synthesis after the G1 block. PMID: 3056733 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: J Virol. 1988 Mar;62(3):1028-37. Characterization of simian virus 40 large T antigen by using different monoclonal antibodies: T-p53 complexes are preferentially ATPase active and adenylylated. Tack LC, Wright JH, Gurney EG. Molecular Biology and Virology Laboratory, Salk Institute, La Jolla, California 92138. We used 21 monoclonal antibodies (PAbs 100 to 117, 405, 419, and KT3) specific for different determinants in simian virus 40 (SV40) large T antigen (T) and one antibody specific for p53 that coprecipitates T complexed with p53 (T-p53) to analyze T in SV40-infected CV1 cells. We measured the ATPase specific activity, extent of adenylylation, and p53 content of T precipitated by antibodies directed against the N-terminal region I (0.65 to 0.62 map units), the midregion III (0.43 to 0.28 map units) containing both the ATPase- and nucleotide-binding sites, and the C-terminal region IV (0.28 to 0.17 map units) of T. Lytic T appeared to exist in three different forms with respect to p53 binding and ATPase activity. The most ATPase-active form of T was that precipitated by PAb 122. This T-p53 complex contained only 6% of the total T but contributed 35% of the ATPase activity, on average. Free p53 isolated from 3T6, Ann-1, or L929 cells had no apparent ATPase activity. A second form of T precipitated by several antibodies had little associated p53 but appreciable ATPase activity, accounting for 15 to 20% of total T and 60 to 70% of the ATPase activity. The rest of T constituted the third form and was also depleted in p53 but had a decreased ATPase specific activity. Thus, the remaining 75 to 80% of T had 15 to 20% of the ATPase specific activity. Antibodies specific for region III precipitated T with both altered ATPase activity and altered amounts of bound p53. PAbs 104 and 114 reacted with ATPase-active T but inhibited ADP hydrolysis, suggesting that they were inactivating antibodies. T that was preferentially adenylylated in vitro corresponded to T that was also preferentially ATPase active. T bound to p53 was adenylylated to a higher specific activity than total T. In addition, p53 itself was significantly adenylylated under these conditions. The results suggest that ATPase activity and p53 binding are structurally and functionally related and that p53 alters biochemical activities of T and plays a role in productive infection. PMID: 2448496 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------