Lymphoid cells and small intestinal epithelial (SIE) cells are among the most radiosensitive in the body. The factors that account for the differential sensitivity to gamma-radiation among different tissue-types remain poorly understood, but can only partly be explained by differences in rates of cell proliferation. Here we demonstrate that exposure of mice to 800 cGy of gamma-radiation results in rapid elevations in the levels of the Bax protein, a pro-apoptotic member of the Bcl-2 protein family, in lymphoid cells and SIEs. gamma-Radiation-induced increases in Bax protein were evident within 2 h and persisted for at least 24 h, as determined by immunoblotting and immunohistochemical assays. Increases in Bax were followed by massive apoptosis in lymphoid organs and in the small intestinal crypts, as determined by morphological criteria and in situ end-labeling of fragmented nuclear DNA by terminal deoxynucleotidyl transferase (TUNEL method). Radiation did not induce elevations in Bax or apoptosis in radioresistant tissues such as heart, skeletal muscle, brain, kidney, liver, lung, vascular smooth muscle and connective tissue. The findings suggest that Bax may be one of the mediators of radiation-induced apoptosis in vivo.

The bcl-2 gene becomes activated in many types of human cancers and contributes to neoplastic cell expansion, as well as to resistance to radiation and chemotherapy, by blocking programmed cell death or apoptosis. The expression of this proto-oncogene is regulated at both the transcriptional and post-transcriptional levels. DNA sequence comparisons of human, mouse, rat and chicken bcl-2 cDNAs revealed the presence of an open reading frame (ORF) [correction of (OFR)] located upstream of the normal coding region. Because upstream ORFs (uORFs) have been associated with translational repression, we analysed the functional significance of the 11 amino-acid uORF in the human BCL-2 gene (-119 to -84 bp). Deletion of this uORF from chloramphenicol acetyltransferase (CAT) reporter gene constructs that contained the bcl-2 promoter and entire 5'-untranslated region (5'-UTR), as well as introduction of an A-->T mutation at position -119 bp that destroyed the AUG-initiation codon, significantly increased CAT activity in HeLa, CEM, and other cell lines, without producing a corresponding elevation in CAT mRNA levels. Positioning this uORF, together with its accompanying Kozak sequences, between a heterologous promoter from SV40 and a CAT reporter gene resulted in marked inhibition of CAT protein production without a decrease in CAT mRNA. Mutation of the start codon (ATG-->TTG) of this uORF completely abolished its inhibitory activity, consistent with a translational mechanism. Taken together, these findings suggest that the uORF located within the 5'UTR of the bcl-2 gene is necessary and sufficient for translational regulation of bcl-2 gene expression.

P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.

The cancer chemopreventive retinoid N-(4-hydroxyphenyl)-all-trans retinamide (HPR) was recently shown by us to have antiproliferative and apoptotic effects on human leukemic cell lines, including those unresponsive to all-trans retinoic acid (ATRA). We have now characterized further the process of HPR-induced cell death. We report that inhibitors of RNA transcription and of protein synthesis, activators of protein kinase C (PKC), inhibitors of tyrosine kinases, Zn++, and the antioxidants acetylcysteine, ascorbic acid, alpha- tocopherol, and deferoxamine suppressed HPR-induced apoptosis. HL60 cells induced toward monocytic differentiation by 1,25 dihydroxyvitamin- D3 [1,25(OH)2D3], but not those induced toward the granulocytic differentiation by ATRA, showed reduced responses to HPR. The transport of HPR by cells with different sensitivity to the retinoid, however, was similar, even after treatment with the phorbol ester 12-O- tetradecanoylphorbol-13-acetate (TPA), which induces unresponsiveness to HPR. The expression of the apoptosis-related genes bcl-2, p53, and c- myc was examined to determine their role in HPR-triggered cell death. The levels of bcl-2 mRNA were markedly diminished by 24 hours of HPR treatment in all cell lines except in the relatively HPR-insensitive line K422. However, probably because of its long half-life, bcl-2 protein levels were either unchanged or only slightly decreased. Downregulation of p53 mRNA was also observed within 24 hours of HPR exposure in NB4 but not K422 cells, but no changes in the amount of p53 protein were found. Suppression of c-myc transcription was observed in all cells except K422. The protective role of bcl-2 on cell death by HPR was investigated in HL60 as well as 697 pre-B leukemia and Jurkat T- acute lymphocytic leukemia (T-ALL) cells constitutively expressing high levels of bcl-2 proteins due to gene transfer manipulation. Compared with control cells, the onset of apoptosis in these cells with deregulated bcl-2 production was delayed by at least 24 hours. These findings establish that cell death by HPR requires RNA transcription and protein synthesis and is regulated by the activation of PKC. Although changes in bcl-2, p53, and c-myc expression are found in cells treated with HPR, the time-course of these events suggests that HPR- triggered apoptosis is not directly controlled by these genes. Finally, while ectopic overexpression of bcl-2 does not protect cells from death by HPR, it markedly delays its onset.(ABSTRACT TRUNCATED AT 400 WORDS)

The bcl-2 gene becomes dysregulated in its expression in a wide variety of human cancers and has been shown to block both spontaneous and drug-induced cell death, thus conferring a selective survival advantage on malignant cells. The biochemical mechanism by which bcl-2 promotes cell survival remains enigmatic but appears to involve a downstream event in an evolutionarily conserved cell death pathway. Here we report that gene transfer-mediated increases in Bcl-2 protein levels in the human leukemia line Jurkat render these cells more resistant to induction of DNA fragmentation and cytolysis by a cloned T-cell. The killing mechanism used by these particular T-cells was consistent with apoptosis, as opposed to necrosis, in that DNA degradation occurred as a prelysis event. The findings raise the possibility that dysregulation of bcl-2 gene expression could play a role in the avoidance of immune surveillance mechanisms by cancer cells.

Recently, both Bcl-2, which promotes cell survival, and Bax, which promotes cell death, have been implicated as major players in the control of apoptotic pathways, and it has been suggested that the ratio of Bcl-2 and Bax protein controls the relative susceptibility of cells to death stimuli. We have used M1 myeloid leukemia cells and genetically engineered M1 variants as a model system to study apoptosis induced by two distinct apoptotic stimuli. This includes apoptosis induced by activation of wild type p53 function of a temperature sensitive p53 transgene expressed in M1 cells, which do not express endogenous p53, and apoptosis induced by TGF beta 1. It is shown that the kinetics of apoptosis induced by p53 is more rapid than apoptosis induced by TGF beta 1. It is also shown that ectopic expression of Bcl-2, at levels which blocked TGF beta 1-induced apoptosis of M1 cells, delayed, but did not block, p53-induced apoptosis. Both p53 and TGF beta 1 down-regulated endogenous Bcl-2 expression, but only p53 up-regulated Bax expression, where bax has been identified as a p53 immediate early response gene. Thus, the p53-mediated up-regulation of Bax may provide at least a partial explanation for the more rapid rate of apoptosis induced by p53 compared to by TGF beta 1, as well as for the ineffectiveness of ectopoic Bcl-2 to abrogate p53-mediated apoptosis. These findings provide first insights to the molecular mechanisms which mediate p53-induced apoptosis, identifying bax and bcl-2 as p53 regulated genes, and serve as a paradigm of how the intracellular balance of Bcl-2 to Bax is differentially altered by distinct death stimuli.

The Bcl-2 protein is over-produced in many types of human tumors and suppresses apoptosis induced by a wide-variety of stimuli, including chemotherapeutic drugs and gamma-irradiation. The biochemical mechanism of action of the Bcl-2 protein however remains enigmatic. Here we show that Bcl-2 can be co-immunoprecipitated with the serine/threonine-specific Raf-1 kinase both in a mammalian hemopoietic cell 32D.3 and when the two proteins are produced in Sf9 insect cells using recombinant baculoviruses. Though analysis of Raf-1 deletion mutants suggested that the C-terminal half of the protein which contains the catalytic domain is sufficient for co-immunoprecipitation with Bcl-2, Raf-1 does not appear to induce phosphorylation of Bcl-2 protein in 32D.3 and Sf9 cells. Furthermore, a mutant form of Raf-1 that lacks kinase activity could still be co-immunoprecipitated with Bcl-2 in Sf9 cells, suggesting that the interaction of these proteins does not reflect a kinase-substrate relation. Gene transfer experiments using 32D.3 hemopoietic cells demonstrated functional synergy between Bcl-2 and Raf-1 with regards to suppression of apoptosis induced by growth factor withdrawal. Taken together, these observations for the first time functionally link Bcl-2 to a signal transducing protein and suggest that the interaction of the Bcl-2 and Raf-1 proteins may be responsible for their ability to cooperate in the suppression of apoptosis.

The protein encoded by the bcl-2 gene is a regulator of programmed cell death and apoptosis. The cell survival-promoting activity of this protein is opposed by Bax, a homologous protein that forms heterodimers with Bcl-2 and accelerates rates of cell death. In this report, the in vivo patterns of bax gene expression were immunohistochemically assessed in the mouse, with a polyclonal antibody raised against a synthetic peptide corresponding to a unique region in the murine Bax protein. Direct comparisons were made with Bcl-2 by using anti-peptide antisera specific for the mouse Bcl-2 protein. The expression of bax was more widespread than bcl-2. For example, Bax immunoreactivity was present in the hepatocytes of the liver, the exocrine pancreas, and the renal tubule epithelial cells whereas Bcl-2 was absent from these tissues. Both the Bax and Bcl-2 proteins were present in several epithelia examined, including the small intestines, colon, breast, prostate, respiratory tract, and skin. The most intense Bax immunostaining was seen in cells located in the base of the crypts of the small intestinal mucosa, consistent with reports of high rates of spontaneous and inducible apoptosis in this region. Bcl-2 immunostaining was completely absent from these cells but was present in the absorptive epithelial cells of the small intestine. In contrast, Bax immunostaining in the colon tended to be stronger in the surface epithelial cells that had advanced up the crypts towards the lumen and that are destined for programmed cell death, whereas Bcl-2 immunoreactivity generally was stronger in the base of the colonic crypts. Similarly, bax expression in the gastric pits of the stomach occurred in a gradient such that higher levels of Bax immunostaining were found in the upper layers of gastric glands than in the lower regions. In addition, strong Bax immunostaining was detected in the androgen-dependent secretory epithelial cells of the prostate, whereas Bcl-2 was limited to the androgen-independent basal cells. Like Bcl-2, Bax was found in the thymic medulla but not the cortex, despite the propensity for immature cortical thymocytes to undergo apoptosis. Unlike Bcl-2, however, Bax immunostaining tended to be more intense in the germinal center lymphocytes of lymph nodes than in the interfollicular lymphocytes, consistent with the high rate of apoptotic cell death in the former.(ABSTRACT TRUNCATED AT 400 WORDS)

Recently, we have shown that the p53 tumor suppressor gene product can inhibit expression of the bcl-2 gene. In this report, we explored the molecular basis for p53-mediated down-regulation of bcl-2 gene expression using a cotransfection approach involving p53 expression plasmids and chloramphenicol acetyltransferase (CAT) reporter gene constructs containing regions from the bcl-2 gene. When transfected into a p53-deficient human lung cancer cell line H358, reporter gene constructs containing only the promoter region of bcl-2 and upstream sequences were not suppressed by p53. Inclusion of bcl-2 gene sequences corresponding to the 5' untranslated region in bcl-2/CAT constructs, however, resulted in p53-dependent down-regulation. A 195-base pair segment from the bcl-2 gene 5' untranslated region was found to be capable of conferring p53-dependent repression on a heterologous expression plasmid containing CAT under the control of an SV40 immediate early-region promoter. This p53-negative response element functioned in an orientation-independent manner when placed either upstream or downstream of the SV40-CAT transcription unit. The results demonstrate the existence of a negative response element in the bcl-2 gene through which p53 may either directly or indirectly transcriptionally down-regulate expression of this gene involved in the regulation of programmed cell death.

BACKGROUND:The bcl-2 gene becomes activated by 14;18 chromosomal translocations in the majority of low-grade non-Hodgkin's lymphomas (NHLs) and is expressed at high levels in the absence of gene rearrangements in a high proportion of B-cell chronic lymphocytic leukemias (B-CLLs). The protein encoded by bcl-2 contributes to neoplastic cell expansion by prolonging cell survival through its ability to block programmed cell death (apoptosis). Because many chemotherapeutic drugs have been shown ultimately to kill tumor cells through mechanisms consistent with programmed cell death, we tested whether the relative levels of bcl-2 oncoprotein influence the sensitivity of lymphoma and leukemia cell lines to killing by conventional cytotoxic drugs commonly used in the treatment of cancer.METHODS:Leukemia cell lines with low levels of bcl-2 expression were stably infected with recombinant bcl-2 retroviruses to achieve elevations in bcl-2 protein levels. Lymphoma cell lines with high levels of bcl-2 expression as the result of 14;18 translocations were either stably transfected with inducible bcl-2 antisense expression plasmids or treated with bcl-2 antisense oligonucleotides to achieve reductions in bcl-2 protein levels. The sensitivity of these genetically modified cells to killing by various antineoplastic drugs was then determined.RESULTS:Gene transfer-mediated elevations in bcl-2 protein levels in lymphocytic leukemia cell lines was correlated with markedly elevated resistance to killing by all cytotoxic drugs tested. Conversely, antisense-mediated reductions in bcl-2 protein levels in t(14;18)-containing NHL cell lines resulted in enhanced sensitivity to all anticancer drugs.CONCLUSIONS:The relative levels of bcl-2 oncoprotein represent one of the key determinants of the sensitivity of lymphocytic cells to killing by essentially all drugs currently available for the treatment of cancer.

The p53 tumor suppressor gene product can induce apoptotic cell death through an unknown mechanism. Here we demonstrate that a temperature-sensitive p53 induces temperature-dependent decreases in the expression of the apoptosis-suppressing gene bcl-2 in the murine leukemia cell M1, while simultaneously stimulating increases in the expression of bax, a gene which encodes a dominant-inhibitor of the Bcl-2 protein. Mice deficient in p53 exhibit increases in Bcl-2 and decreases in Bax protein levels in several tissues as determined by immunohistochemical and immunoblot methods. The findings suggest a potential mechanism by which p53 regulates apoptosis, as well as responses to radiation and chemotherapeutic drugs in cancer.

The bcl-2 gene was originally cloned because of its involvement in B-cell lymphomas and encodes a 25-kD integral membrane protein that has been shown to inhibit programmed cell death (also termed apoptosis) in a wide variety of circumstances. The Epstein-Barr Virus (EBV) also has been implicated in B-cell malignancies and interestingly contains an open reading frame (BHRF-1) predicting a 19-kD protein with 22% homology to Bcl-2. To compare the functions of p26-Bcl-2 and p19-BHRF-1, we stably introduced expression plasmids encoding these proteins into a murine interleukin-3 (IL-3)-dependent hemopoietic cell line, 32D. Removal of IL-3 from cultures of control-transfected 32D cells resulted in internucleosomal DNA cleavage (a hallmark of programmed cell death) and loss of cell survival. In contrast, 32D cells containing high levels of p26-Bcl-2 or p19-BHRF-2 proteins exhibited prolonged survival and markedly delayed DNA degradation under the same conditions of IL-3 deprivation. As a first attempt to determine the functional importance of amino acid sequences that are conserved between the Bcl-2 and BHRF-1 proteins, we used site-specific mutagenesis to replace two conserved cysteine residues with alanines (positions 158 and 219) in the human Bcl-2 protein. Comparisons of the wild-type and cysteine-minus human Bcl-2 proteins in S49 lymphoma cells revealed equivalent ability to block glucocorticoid-induced cell death and DNA fragmentation, indicating that these two conserved cysteines are not critical for Bcl-2 oncoprotein function. Investigations in 32D cells of an avian homolog of Bcl-2 cloned from the chicken also revealed conservation of function with the human Bcl-2 protein, despite the presence of a 48-amino-acid region of divergent sequence. Taken together, these data demonstrate that despite marked differences in their predicted amino-acid sequences, the human, chicken, and EBV versions of Bcl-2 have retained the structural characteristics necessary to interface with pathways involved in the regulation of programmed cell death in murine cells. The findings thus contribute to the mapping of functional domains in Bcl-2 proteins, and raise the possibility that the EBV-encoded p19-BHRF-1 protein may be able to substitute for p26-Bcl-2 in the development of some types of cancer.

Previous studies have shown that the bcl-2 gene encodes a mitochondrial protein that contributes to neoplastic cell expansion primarily by promoting cell survival through interference with “programmed cell death” (PCD), also termed “apoptosis.” Because many chemotherapeutic drugs are capable of initiating pathways leading to apoptosis, we determined whether deregulated bcl-2 expression could render cells resistant to several drugs commonly used in the treatment of non- Hodgkin's lymphomas, including dexamethasone (DEX), methotrexate (MTX), 1-beta-D-arabinofuranosyl-cytosine (Ara-C), etoposide (VP-16), vincristine (VC), cisplatin (CP), and hydroperoxycyclophosphamide (4- HC). For these experiments, we achieved high levels of p26-Bcl-2 protein production in a human pre-B-cell leukemia line 697 by stable infection with a recombinant bcl-2-containing retrovirus and then compared these cells with control virus-infected 697 cells. Control 697 cells were induced to undergo apoptosis by all drugs tested as defined by DNA degradation into oligonucleosomal-length fragments, cell shrinkage, and subsequent cell death. In contrast, 697 cells with elevated Bcl-2 protein levels exhibited strikingly prolonged cell survival and markedly reduced DNA fragmentation when cultured in the presence of these antineoplastic agents. Although high levels of Bcl-2 protein protected 697 cells from the acute cytotoxic effects of DEX and the other drugs tested, Bcl-2 did not prevent these drugs from suppressing the proliferation of 697 cells. However, when 697 cells were treated with DEX or MTX for 3 days, then washed and cultured in semisolid media without drugs, bcl-2-virus-infected cells gave rise to colonies at much higher frequencies than 697 cells stably infected with control virus. These results indicate that by protecting 697 leukemic cells from the acute cytotoxicity of DEX and some other chemotherapeutic drugs, high levels of p26-Bcl-2 can create the opportunity for re-initiation of cell growth when drugs are withdrawn. The findings may be relevant to clinical correlative studies of non- Hodgkin's lymphoma patients that have found an association between worse prognosis and bcl-2 gene rearrangements or t[14;18] translocations.