Toshiyuki Miyashita

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.

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.

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.

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)