Lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in testicular germ cell tumour cell lines

We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat.     

Old and new substrates in clinical nutrition

Bcl-2 has been demonstrated to inhibit apoptosis in breast cancer cells in vitro, and the ratio between Bcl-2 and its proapoptotic homologue Bax seems to be an important determinant of cellular sensitivity to induction of apoptosis. However, little information is available on the relationship between Bcl-2 and the rate of apoptotic and necrotic cell death in breast tumours. From a series of 441 premenopausal, lymphnode-negative breast cancer patients, a subset of 49 tumours was selected in which immunostaining for the 26-kDa isoform of Bcl-2 was either absent (n = 23) or very high (n = 26). High expression of Bcl-2 was found to be strongly associated with low rates of apoptotic (P < 0.001) and necrotic cell death (P < 0.001). The mean value of the apoptotic index was 2.69%+/-1.40% in Bcl-2-negative tumours and 0.68%+/-1.00% in Bcl-2-positive tumours. Expression of the proapoptotic protein Bax correlated neither with Bcl-2 nor with the frequency of apoptotic cells. Immunostaining for the antiapoptotic Bcl-2 homologue BcI-X(L) correlated with Bcl-2 expression (P < 0.001) but not with apoptosis. High proliferation rate and high tumour grade (Bloom-Richardson) were strongly associated with absence of Bcl-2 expression (P< 0.001). p53 accumulation was associated with absence of Bcl-2 expression and increased apoptotic activity. Loss of Bcl-2 expression was strongly correlated with increased apoptotic and necrotic cell death, high proliferation rate and high tumour grade, supporting a model in which Bcl-2 not only mediates cell death, but also cell division in breast cancer tissue, and in which regulation of cell division and cell death are tightly linked.     

Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Apoptosis-related factors p53, Bcl2, and Bax in neuroendocrine lung tumors

Neuroendocrine (NE) lung tumors comprise four classes of progressive aggressiveness for which proliferation and apoptosis rates could both contribute to their distinctive behavior. As p53 mutations may favor escape from apoptosis through changes in Bcl2-Bax expression balance, which are survival and apoptotic genes, respectively, we studied 121 NE lung tumors (16 typical carcinoids (TC), 5 atypical carcinoids (AC), 29 large-cell NE carcinomas (LCNECs), and 71 small-cell lung carcinomas (SCLCs) using immunohistochemistry. We quantified apoptosis by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) in 31 of these cases. There was a significant increase of p53 mutant immunophenotype (defined as immunoreactivity with at least two antibodies for at least 20% of tumor cells) between atypical/typical carcinoids group and the LCNEC/SCLC group (P = 0.0003). There was an inverse correlation (P < 0.0001) between the scores of Bax and Bcl2 expression in individual tumors and a significant inversion of the Bcl2. Bax ratio between low-grade (typical and atypical carcinoids) and high-grade (LCNECs and SCLCs) tumors with a predominant Bax expression in the first group and predominant Bcl2 expression in the second. Whereas carcinoids had variable apoptotic indexes, LCNECs had high indexes (1.3 to 6.8%), Bcl2 overexpression, Bax down-regulation, and Bcl2.Bax ratio > 1 correlated with lower apoptotic index in both LCNEC and the pool of LCNECs and SCLCs (P < 0.05) and a lower survival rate in the group of atypical and typical carcinoids and LCNECs (P < 0.002). The highest levels of Bcl2 expression and Bcl2.Bax ratios were associated with p53 mutant immunophenotype (P = 0.02). Our results suggest that aggressiveness in NE lung tumors could be linked, in addition to proliferation, to apoptosis-related factors.     

Expression of p53 protein has no independent prognostic value in breast cancer

A series of 392 female breast carcinomas was analysed immunohistochemically for expression of p53 protein with special emphasis on the role of p53 as an independent prognostic factor. Altogether, 54.8 per cent of the carcinomas expressed p53 protein, with the mean [standard error (SE)] fraction of positive nuclei being 17.1 per cent (1.2 per cent). Expression of p53 protein was independent of tumour metastasis at diagnosis, axillary lymph node status, tumour diameter, histological type, tubule formation, proportion of intraductal growth, margin formation, necrosis, DNA ploidy, and S-phase fraction. A high fraction of p53-positive nuclei was significantly related to patient age under 70 years, high grade, severe nuclear pleomorphism, dense infiltration of tumour by lymphocytes, high mitotic index, and high apoptotic index (for all, P < 0.05). Impaired survival probability in the entire cohort (P = 0.05) and in the axillary lymph node-positive (ANP) tumours (P = 0.015) was associated with a fraction of p53-positive nuclei less than 25 per cent, while in the axillary lymph node-negative (ANN) tumours, expression of p53 had no prognostic value. In multivariate analysis, independent prognostic predictors included axillary lymph node status, tumour diameter, and mitotic index. In the ANN tumours, tumour diameter, fraction of p53-positive nuclei, and tumour grade were independent prognostic factors, whereas in the ANP tumours, diameter and mitotic index were the two independent prognostic factors. The results suggest that abnormal expression of p53 protein is only a weak independent prognostic factor in female breast cancer.     

Ochratoxin A disturbs pH homeostasis in the kidney: increases in pH and HCO3- in the tubules and vasa recta

The aim of this study was to ascertain whether apoptotic counts have prognostic significance in colorectal cancer and if such counts are related to the expression of proteins implicated in cell cycle regulation. Material from a cohort of patients aged 45 years or less with colorectal carcinoma was re-examined to determine apoptotic and mitotic counts by light microscopy, in addition to assessing p53, c-myc, and bcl-2 protein status by immunohistochemistry. The apoptotic index in the 74 patients who were alive or who had died of colorectal carcinoma ranged from 1.2 per cent to 12.3 per cent and exhibited independent prognostic significance, with high counts predicting better survival (P = 0.02). Mitotic counts were not related to survival, despite a close correlation with apoptosis (r = 0.85). Tumours regarded as not staining with the CM1 antibody for p53 protein demonstrated higher apoptotic counts, compared with those that stained (medians 5.2 and 4.0 per cent, respectively; P = 0.03), but p53 expression was found not to be related to survival. The 68 tumours which stained for c-myc appeared to exhibit higher mitotic counts than those that did not. bcl-2 was detected in only four tumours. The latter two proteins exhibited no apparent relationship to the apoptotic index or survival. Although these results indicate a potential role for apoptotic counting in prognostic prediction in colorectal tumours, this is an uncommon group of patients who exhibited some atypical features. The likelihood of a proportion of cases arising within hereditary non-polyposis colorectal cancer syndrome may limit the application of the findings to a more general population with cancer of the colon and rectum. Further work is required, including critical measurement of reproducibility and assessment of the relative impact of this parameter compared with 'traditional' prognostic markers.     

UCN-01 in ovary cancer cells: effective as a single agent and in combination with cis-diamminedichloroplatinum(II)independent of p53 status

Our goal was to determine the cytotoxicity of 7-OH-hydroxystaurosporine (UCN-01) as a single agent and in combination with cis-diamminedichloroplatinum(II) (CDDP) in a panel of ovarian carcinoma cells. We sought to examine the role of p53 gene function and alterations in cell cycle progression or other mechanisms of action of UCN-01 including perturbation of the apoptosis pathway mediated by NF-kappaB and Bcl-2/Bax. Cytotoxicity was determined from dose-response curves established by the Alamar blue vital dye indicator assay. Restoration of wild-type p53 in a p53 null cell line, SKOV 3, was achieved by transfection of a p53 expression vector. Cell cycle distribution was measured by fluorescence-activated cell sorting analysis of ethidium bromide-stained nuclei. Apoptosis was measured by quantitative fluorescence microscopy. NF-kappaB DNA binding activity was measured by electrophoretic mobility shift assay. Bcl-2 and Bax levels were determined by Western immunoblotting. UCN-01 was effective as a cytotoxic agent alone and in combination with CDDP in all cell lines studied, regardless of p53 status. The degree of sensitization to CDDP conferred by UCN-01, however, was found to correlate with p53 gene status. p53 wild-type cells seem to be more sensitive to the cytotoxic effects of the combination of UCN-01 + CDDP than the p53 mutant cells. This was confirmed in cells in which p53 wild-type function was restored by transfection of p53 cDNA, but these cells are also significantly more sensitive to CDDP alone. The effects of UCN-01 on cell cycle progression also appear to be p53 dependent but may not be the primary mechanism of action. The rate of apoptosis is increased 4-fold in UCN-01 + CDDP-treated cells compared to either agent alone. UCN-01 does not effect NF-kappaB DNA binding activity or Bcl-2 and Bax levels. UCN-01 enhances CDDP cytotoxicity and apoptosis in ovary cancer cells. This occurs regardless of p53 status, but wild-type p53 seems to increase the degree of sensitization.     

Protective effects of the antiparkinsonian drugs talipexole and pramipexole against 1-methyl-4-phenylpyridinium-induced apoptotic death in human neuroblastoma SH-SY5Y cells

Treatment of human neuroblastoma SH-SY5Y cells with 1 mM 1-methyl-4-phenylpyridinium (MPP+) for 3 days induced production of reactive oxygen species (ROS), followed by caspase-3 activation, cleavage of poly(ADP-ribose) polymerase (PARP), and apoptotic cell death with DNA fragmentation and characteristic morphological changes (condensed chromatin and fragmented nuclei). Simultaneous treatment with 1 mM talipexole slightly inhibited the MPP+-induced ROS production and apoptotic cell death. In contrast, pretreatment with 1 mM talipexole for 4 days markedly protected the cells against MPP+-induced apoptosis. However, this protective effect might not be mediated by dopamine receptors. The talipexole pretreatment induced an increase in antiapoptotic Bcl-2 protein level but had no effect on levels of proapoptotic Bax, Bak, and Bad. It also inhibited MPP+-induced ROS production, p53 expression, and cleavages of caspase-3 and PARP. Similarly, pramipexole pretreatment increased Bcl-2 and inhibited MPP+-induced apoptosis. Although pretreatment with bromocriptine also had a protective effect against MPP+-induced apoptosis, it had no effect on the protein levels of Bcl-2 family members. On the other hand, N6,2'-O-dibutyryl cAMP or calphostin C induced a decreased Bcl-2 level and enhanced MPP+-induced cell death. These results suggest that talipexole has dual actions: (1) it directly scavenges ROS, affording slight protection against MPP+-induced apoptosis, and (2) it induces Bcl-2 expression, thereby affording more potent protection, if it is administrated before MPP+. Pramipexole has similar effects, whereas bromocriptine seems to exhibit the former but not the latter effect.     

Melatonin chimeras alter reproductive development and photorefractoriness in Siberian hamsters

Apoptosis is well accepted as a type of cell death occurring in the development of mammalian muscles, but the death of adult myofibres in neuromuscular disorders and exercise-induced muscle damage is usually explained in terms of muscle necrosis. The current view that apoptosis precedes necrosis in death of dystrophin-deficient muscle fibres of mdx mouse has been well substantiated. Moreover, apoptotic myonuclei have been reported to increase in mdx mice 2 days after spontaneous exercise. To investigate the contribution of apoptosis to exercise-induced damage of normal muscle fibre a time-course analysis has been performed in adult C57BL/6 mice. Groups of five mice were sacrificed immediately after the end of the exercise, and after a rest period of 6 or 96 h. The amount of apoptosis in leg muscles was assessed by electron microscopy, by the terminal deoxynucleotidyl transferase assay and by electrophoretic detection of fragmented DNA; the expression of Bcl-2, Bax, Fas, ICE, p53 and ubiquitin was examined by immunohistochemistry and Western blot. Absent in muscles of normal 'sedentary' mice, apoptotic myonuclei peak in muscles of normal mice after a night of spontaneous wheel-running (4% +/- 3.5, immediately and 2.5% +/- 1.8 after 6 h rest, P < 0.05 vs non-runner mice); they then decrease but are present 4 days later (0.8% +/- 1.5). Satellite cells are also involved in the apoptotic process. Myofibre content of Bcl-2 decreases whereas Bax, Fas, ICE and ubiquitin modify their pattern of expression in correlation with the changes in apoptotic myonuclei. Apoptosis of endothelial cells is present after the night of wheel-running and with a twofold increase 4 days later (1.5 +/- 2.3 and 4.8 +/- 4.4 P < 0.05, respectively). Satellite cells are also involved in the apoptotic process. Thus, spontaneous running in unaccustomed mice increases the number of apoptotic nuclei in adult muscle fibres and in endothelial cells. It remains to be established whether muscle apoptosis is restricted to the repair mechanisms, as often suggested in many pathologic processes, or it is also part of pathogenesis of muscle damage. Regardless of whether these results are extended to human dystrophies, the clinical implications in terms of secondary pathogenetic mechanisms and muscle training are obvious.     

Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats

Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20+/-0.05; 12 months, 7.21+/-0.07; 24 months, 7.18+/-0.09. In contrast, diastolic Ca2+ determined by the Fura 2-AM method increased progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02). Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.     

Extended survivability of prostate cancer cells in the absence of trophic factors: increased proliferation, evasion of apoptosis, and the role of apoptosis proteins

This project was undertaken to study the survival properties of various prostate cells, including normal (NHP), BPH (benign prostate hyperplasia), primary carcinoma (PCA), and metastatic prostate cancer cells (LNCaP, PC3, and Du145), in the absence of trophic factors. Cell proliferation and cell death were quantitated by enumerating the number of live cells using MTS/PMS kit and of dead (apoptotic) cells using 4',6-diamidino-2-phenylindole dihydrochloride nuclear staining. These cells demonstrated an overall survivability in the order of BPH < NHP < LNCaP < PC3 < PCA < Du145. Upon growth factor deprivation, NHP/BPH cells rapidly underwent apoptosis, leading to a decreased number of live cells. PCA/PC3/Du145 cells, in contrast, demonstrated an initial phase of aggressive growth during which apoptosis rarely occurred, followed by a "plateau" phase in which cell loss by apoptosis was compensated by cell proliferation, followed by a later phase in which apoptosis exceeded the cell proliferation. LNCaP cells demonstrated survival characteristics between those of NHP/BPH and PCA/PC3/Du145 cells. We concluded that the increased survivability in prostate cancer cells results from enhanced cell proliferation as well as decreased apoptosis. The molecular mechanisms for evasion of apoptosis in prostate cancer cells were subsequently investigated. Quantitative Western blotting was used to examine the protein expression of P53 and P21WAF-1, Bcl-2 and Bcl-X(L) (anti-apoptotic proteins), and Bax, Bak, and Bad (proapoptotic proteins). The results revealed that, upon trophic factor withdrawal, NHP and BPH cells upregulated wild-type p53 and proapoptotic proteins Bax/Bad/Bak and down-regulated the expression of P21. Furthermore, NHP and BPH cells endogenously expressed little or no Bcl-2. In sharp contrast, prostate cancer cells expressed nonfunctional P53 and various amounts of Bcl-2 proteins. Upon deprivation, these cancer cells up-regulated P21 and Bcl-2 and/or BclX(L), lost response to withdrawal-induced up-regulation of Bax/Bad/Bak or decreased or even completely lost Bax expression and expressed some novel proteins such as P25 and P54/55 complex. These data together suggest that prostate cancer cells may use multiple molecular mechanisms to evade apoptosis, which, together with increased proliferation, contribute to extended survivability of prostate cancer cells in the absence trophic factors.     

The rhesus monkey analogue of human lymphocyte antigen-G is expressed primarily in villous syncytiotrophoblasts

The human placenta expresses the nonclassical major histo-compatibility complex (MHC) class I molecule, human lymphocyte antigen (HLA)-G, which may contribute to the establishment of maternal-fetal immune tolerance. Although the HLA-G ortholog of the rhesus monkey, Mamu-G, is a pseudogene, another nonclassical MHC class I locus, Mamu-AG, is expressed in the rhesus monkey placenta. Mamu-AG encodes MHC class I A locus-related molecules that exhibit all the characteristics of human HLA-G, including limited polymorphism and a truncated cytoplasmic domain. We have examined MHC class I glycoprotein and Mamu-AG mRNA expression in the rhesus placenta and in cultured trophoblasts. Immunocytochemical analysis of rhesus placental tissues with the W6/32 monoclonal antibody demonstrated a high level of MHC class I expression in villous syncytiotrophoblasts, whereas villous cytotrophoblasts were largely MHC class I negative. Only low levels of MHC class I expression were seen in extravillous cytotrophoblasts of cell columns and the trophoblastic shell. In situ hybridization demonstrated that Mamu-AG mRNAs were expressed at a high level in first-trimester villous syncytiotrophoblasts. MHC class I and Mamu-AG expression was significantly up-regulated during in vitro culture and differentiation of freshly isolated villous cytotrophoblasts into syncytiotrophoblasts. Preferential Mamu-AG expression in syncytiotrophoblasts suggests that rhesus monkey MHC class I-bearing trophoblasts could potentially interact with maternal peripheral blood lymphocytes rather than with uterine decidual lymphocytes as has been proposed for human trophoblasts.     

Differential expression of cell survival and cell cycle regulatory proteins in cutaneous squamoproliferative lesions

Previous models of cutaneous carcinogenesis have primarily focused on the regulation of keratinocyte (KC) proliferation and differentiation. However, it has become clear in many neoplastic systems that altered rates of cell death and/or inability to undergo growth arrest can also contribute to the development of cancer. Apoptosis-regulatory proteins include those that block apoptosis such as Bcl-2 and Bcl-x, whilst a related protein Bax promotes apoptosis. Cell cycle regulatory proteins include those associated with growth arrest, i.e. p21wafl, p53, and those associated with proliferation, i.e. Ki-67. Paraffin embedded samples from ten different lesions of squamous cell carcinoma (SCC), Bowen's disease (BD), keratoacanthomas (KA), and nine normal adult skin samples were stained by immunohistochemistry to detect expression of Bcl-2, Bcl-x, Bax, Ki-67, p21wafl, p53 and apoptosis (TUNEL assay). Compared to low levels of Bcl-x and Bcl-2 immunostaining in normal skin, all the squamoproliferative lesions had strong and diffuse KC expression of Bcl-x (>80%) but minimal to absent KC Bcl-2 expression (<15%). Bax immunopositivity was limited to the basal layer in normal skin and BD. In contrast, by examining serial sections both Bcl-x and Bax appeared to be coexpressed by the majority of malignant KCs in KA and SCC (>70%). These immunostaining profiles reveal that squamoproliferative lesions, including invasive transformed KCs, preferentially express Bcl-x over Bcl-2, in addition to upregulating their Bax levels. Even though there were numerous TUNEL positive cells in these squamoproliferative lesions, no other evidence of apoptosis was seen reinforcing the necessity to use caution when relying on TUNEL staining for identification of programmed cell death in skin biopsies. Normal sun-exposed skin had low but detectable p53 and rare p21wafl KC expression. Significantly higher numbers of p21wafl and p53 immunopositive KCs were noted throughout the lesions in BD and SCC in contrast to KA where p53 and rare p21wafl immunopositive KCs were primarily limited to the periphery of the tumor cell islands. In general, p53 KC expression was higher in all squamoproliferative lesions and sun-exposed normal skin compared to p21Wafl expression. Summary of the expression of cell cycle regulatory proteins for both p21wafl and p53 KC expression was: SCC > BD > KA, in marked contrast to Ki-67 KC expression which was: BD > KA > SCC. The relatively few malignant cells in SCC that were actively participating in the cell cycle (i.e. Ki-67 positive) suggests that these neoplasms may arise primarily by increased cell survival and resistance to apoptosis rather than by hyperproliferation. These studies emphasize the importance of examining multiple members of protein families that regulate apoptosis, proliferation, growth arrest, and differentiation. It is the overall balance between these cellular phenomena that determine whether a cell remains viable or undergoes programmed cell death and contributes to the appearance of a neoplasm. The overexpression of Bcl-x may confer a survival advantage to malignant KCs unable to growth arrest to repair damaged DNA (mutant p53) and/or undergo terminal differentiation (increased p21wafl). Thus, mutation or aberrant expression of such proteins may participate in the multistep process of carcinogenesis that gives rise to these squamoproliferative lesions.     

Bcl-2 expression and its association with cell kinetics in human gastric carcinomas and intestinal metaplasia

The bcl-2 protooncogene was initially discovered at the t(14;18) chromosomal breakpoint in follicular lymphomas. It has been demonstrated that bcl-2 protein (Bcl-2) expression blocks apoptosis and plays an important role in cell development and maturation. In the present study, Bcl-2 expression was immunohistochemically examined in 103 cases of gastric carcinoma, as well as 64 cases of non-carcinous gastric mucosa, and its correlation with apoptosis, cell proliferation and p53 immunoreactivity was investigated. Bcl-2 was detected in 18.0% of differentiated-type gastric carcinomas (9 of 50) and 7.5% of the undifferentiated type (4 of 53). In adjacent intestinal metaplastic gastric epithelium, the incidence of Bcl-2 positivity in the incomplete type (21/23, 91.3%) was significantly higher than in the complete type (23/41, 56.1%) (P < 0.04). Double immunostaining for Bcl-2 and Ki-67 clearly revealed the majority of Bcl-2-positive cancer cells to be in a nonproliferating state, although some cancer cells expressed both proteins together. Statistical assessment demonstrated that the average Ki-67 labeling index and apoptotic labeling index in Bcl-2-positive foci were significantly lower than in Bcl-2-negative foci (P < 0.0001, P < 0.0003). In addition, a significant dissociation between Bcl-2 and p53 immunoreactivity was found in cancer tissues. These results indicate that aberrant Bcl-2 expression in gastric carcinomas possibly originates from intestinal metaplastic epithelium, and suggest a possible role in tumor development and growth.     

Regulation of cell survival during B lymphopoiesis: apoptosis and Bcl-2/Bax content of precursor B cells in bone marrow of mice with altered expression of IL-7 and recombinase-activating gene-2

B cell development in mouse bone marrow depends critically upon IL-7. To examine the possible in vivo trophic role of IL-7, we have quantitated apoptosis and Bcl-2 family proteins in populations of phenotypically defined B lineage cells in IL-7-deficient and IL-7-overexpressing mice. Using immunofluorescence labeling, multiparameter flow cytometry, and a short-term culture assay, we show that the apoptotic rates of precursor B cells, but not of more mature B cells, are enhanced by IL-7 gene deletion, associated with increased intracellular content of Bax and decreased Bcl-2, while, conversely, an IL-7 transgene suppresses precursor B cell apoptosis and produces low Bax and high Bcl-2 levels. During normal B cell development, high Bax/Bcl-2 ratios characterize cells undergoing greatest apoptotic cell death. Pro-B cells in RAG-2-/- mice, all destined to abort, show elevated Bax levels and Bax/Bcl-2 ratios. By comparison with the elevated rate of pro-B cell apoptosis in RAG-2-/- mice, provisional estimates have been made for the fraction of pro-B cells undergoing apoptosis in normal mice (70%), IL-7-/- mice (85%), and IL-7 transgenic mice (35%). The results demonstrate that IL-7 strongly promotes in vivo cell survival and maintains antiapoptotic Bcl-2/Bax ratios during the development of precursor B cells in mouse bone marrow.     

Subcallosal striations: early findings of multiple sclerosis on sagittal, thin-section, fast FLAIR MR images

Since Bcl-2 protects a variety of cell types from programmed cell death, whereas Bax promotes apoptosis, the present study examines Bcl-2 and Bax proteins, and bcl-2 and bax mRNA expression in the developing cerebellum of the rat following methylazoxymethanol (MAM) acetate administration by using immunohistochemistry, Western blotting and Northern blotting. Bcl-2 expression in the developing cerebellum is observed in proliferating and differentiating cells, whereas Bax expression is higher in differentiating cells than in proliferating cells during development. Administration of MAM (0.05 microliter/g, i.p.) at postnatal day 3 produces apoptotic cell death, as detected by the characteristic morphology and positivity with the method of in situ end-labeling of nuclear DNA fragmentation of dying cells, in the external granule cell layer of the cerebellum. Dying cells are not stained with Bcl-2 and Bax antibodies. Furthermore, no modification in the intensity of Bcl-2 and Bax protein bands and in the intensity of Bcl-2 and bax mRNA bands on Western and Northern blots, respectively, were observed between control and treated rats. These data indicate that MAM-induced apoptosis is not associated with modifications in the expression of Bcl-2 and Bax.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x(L) and Bcl-x(S), which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma. In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.