There is now good evidence that the cellular protein, p53, is involved in the transformation process, although its precise role is unknown. It was reported recently that expression of the p53 gene can immortalize cells and that the p53 gene can replace the myc oncogene in a myc-ras immortalization/transformation assay. We have investigated whether p53 is involved in the progression towards the neoplastic state in vivo and report here that erythroleukaemic cell lines transformed by different isolates of Friend leukaemia virus show altered expression of the cellular p53 gene. High levels of p53 protein are found in certain lines, but the protein is undetectable in others. This heterogeneity in p53 gene expression is associated with heterogeneity in tumorigenicity. We demonstrate that genomic rearrangements are responsible for p53 gene inactivation in these cell lines and that they occur in vivo during the natural progression of Friend virus-induced erythroleukaemia.

We have studied the role of the excision-repair system and the recombination-repair system in the removal of cross-links and monoadducts caused by furocoumarins plus 360 nm radiation in yeast DNA by neutral and alkaline sucrose gradients and by a fluorometric procedure which detects cross-linked DNA molecules. We found that the excision-repair system, represented by the rad3 mutations, is required both for the removal of monoadducts, causing single-strand break formation, and for the removal of cross-links, causing double-strand break formation. The recombination-repair system, represented by the rad51 mutation, is necessary for double-strand break repair following cross-link removal, but it has no role in the repair of monoadducts. It can be concluded, that at least some of the same enzymes are used in yeast for both the excision of pyrimidine dimers and the excision of cross-links or monoadducts caused by furocoumarins plus light. The RAD3 and RAD51 repair systems, which act independently in the repair of UV-induced lesions, are part of a single system for the repair of cross-links.

A quartz crystal microbalance DNA hybridization biosensor, based on thiol-derivatized peptide nucleic acid (PNA) probes, offers unusual in situ differentiation of single-base mismatches. A large excess of a single-base mismatch oligonucleotide has no effect on the frequency response of the target. Such remarkable distinction between perfect matches and mismatches is illustrated by the detection of a common mutation in the p53 gene. The greater specificity of the new mass-sensitive indicatorless hybridization device over those of analogous PNA-based carbon electrodes is attributed to the formation of a PNA monolayer and the use of a hydrophilic ethylene glycol linker. The improved specificity is coupled to very fast (3-5 min) hybridization in a low-ionic-strength medium.

The p53 gene is rearranged in an erythroleukemic cell line (DP15-2) transformed by Friend retrovirus. Here, we characterize the mutation and identify a deletion of =3.0 kilobases that removes exon 2 coding sequences. The gene is expressed in DP15-2 cells and results in synthesis of a 44,000-dalton protein that is missing the N-terminal amino acid residues of p53. The truncated protein is unusually stable and accumulates to high levels intracellularly. Moreover, it appears to have undergone a change in conformation as revealed by epitope mapping studies. This study represents the first description of an altered p53 gene product arising by mutation during neoplastic progression and identifies a region in the p53 protein molecule that plays a role in determining p53 stability in vivo.There is now good evidence that the cellularly encoded nuclear phosphoprotein, p53, is involved in the transformation process (for reviews, see references 8, 20, and 35). p53 protein levels are elevated in a variety of transformed cells from different species, including cells transformed by viruses (22,23,26,32,43,45) and chemical agents (9). Recently, it was reported that expression of the murine p53 gene can immortalize early-passage rodent cells in culture (17) and that the p53 gene can replace myc in a myc-ras immortalization-transformation assay in rat embryo fibroblasts (12,17,37). Furthermore, overproduction of p53 protein in certain cells has been shown to confer an enhanced tumorigenic phenotype (11,19,32,53). Hence, the p53 gene appears to have oncogenic potential.Several studies indicate that p53 expression is correlated with cell cycling and may play a role in the proliferation of normnal cells (27)(28)(29)(30)40). p53 expression increases before DNA synthesis when resting cells are stimulated to divide by mitogen (30) or by serum (40). In addition, microinjection of p53-specific monoclonal antibodies into the nuclei of quiescent mouse cells blocks their proliferative response to serum stimulation (27,29). Finally, primary cultures of early mouse embryos synthesize p53 (31).Regulation of p53 expression in cells can occur at the level of mRNA abundancy or p53 protein stability (40, 41). Occurrence of the first mechanism was demonstrated in embryonal carcinoma cells (F9) which have nearly 20-foldhigher levels of p53 mRNA than their differentiated progeny (41) and in cells induced to proliferate (30,39,40). In general, however, p53 protein levels are not correlated with the amount of p53 mRNA, indicating that the amount of p53 protein is regulated at the posttranscriptional level, perhaps through changes in protein stability (25,36 the stability and steady-state levels of the p53 protein are considerably increased (36,41). Specific interaction between p53 and the major heat shock proteins HSP68 and HSP70 has also been reported (38).DNA rearrangements have been shown to alter the expression of the p53 gene, leading to complete gene inactivation (32, 54, 55) and expression of truncated proteins antigenically related to p53 (32)....

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Most antitumor agents exert their cytotoxic effect through the induction of apoptosis, and this process may be mediated through an elevation in p53 protein, with a subsequent increase in bax and decrease in bcl-2. p53 also increases mdm-2 expression and mdm-2 may then bind and inactivate p53. Cells from 31 patients with chronic lymphocytic leukemia (CLL) were treated in vitro with 2-chlorodeoxyadenosine (CdA), arabinosyl-2-fluoroadenine (F-ara-A), or chlorambucil (CLB) and drug sensitivity measured using the MTT assay. The protein levels of bax and bcl-2 were measured in CLL cells from 25 patients, and were found to be higher in leukemic cells than in normal B cells. The bcl-2 levels varied three-fold, the bax levels fifteen-fold, and the bax:bcl-2 ratios ranged from 0.44 to 2.91. The expression of mdm-2 mRNA was measured in CLL cells from 28 patients and was found to vary twenty-fold. However, no correlation was observed between drug sensitivity to CdA, F-ara-A, or CLB and the cellular levels of mdm-2 mRNA, or the protein levels of bax or bcl-2, or the bax:bcl-2 ratio. Treatment of CLL cells having wild type p53 with CdA, F-ara-A or CLB produced an increase in p53 protein and mdm-2 mRNA. This was not observed in cells having a p53 mutation, and these cells were highly resistant to both CLB and the nucleoside analogs. In contrast to the nucleoside analogs and CLB, dexamethasone and vincristine had no effect on mdm-2 mRNA levels. Treatment of CLL cells containing a wild type p53 gene with CdA, F-ara-A, or CLB, did not produce any consistent changes in bax or bcl-2. Thus, CdA, F-ara-A and CLB appear to act in CLL cells through a p53-dependent pathway, whereas this does not occur with dexamethasone or vincristine. The cellular levels of mdm-2, bcl-2, bax or the bax:bcl-2 ratios are not predictive indicators of clinical sensitivity in CLL, but an increase in mdm-2 levels after drug treatment is indicative of p53 function in these cells.

BackgroundThe Dlc1 (deleted in liver cancer 1) tumour suppressor gene codes for a RhoGTPase activating protein that is found inactivated in many tumour types. Several transcriptional isoforms have been described but the functional significance and tissue distribution of each form is presently poorly understood. Also, differences in the number of isoforms and splice variants reported still exist between different mammalian species. In order to better understand the number and function of the different variants of the Dlc1 gene in the mouse, we have carried out a detailed analysis. Extensive 3' RACE experiments were carried out in order to identify all possible Dlc1 isoforms and splice variants in the mouse. In addition, we have generated a gene trapped mouse that targets one of these isoforms in order to study its biological function. The effect of this gene trap insertion on the splicing of other isoforms has also been studied.ResultsIn addition to the known 6.1 and 6.2 Kb transcripts of Dlc1, our study revealed the existence of a novel 7.6 Kb transcriptional isoform in the mouse, which corresponds to the human 7.4 Kb (KIAA1723) cDNA transcript. A gene trapped embryonic cell line, with an insertion between Exon 1 and 2 of the 6.1 Kb transcriptional isoform, was used to generate a transgenic mouse. This line showed a significant reduction in the expression of the trapped isoform. However, reduced expression of the other isoforms was not seen. Mice heterozygous for the gene trapped allele were phenotypically normal, but homozygous mutant embryos did not survive beyond 10.5 days post coitum. Dlc1gt/gt embryos showed defects in the brain, heart, and placental blood vessels. Cultured serum-free mouse embryo cells from Dlc1 deficient embryos had elevated RhoA activity and displayed alterations in the organization of actin filaments and focal adhesions. The Dlc1 deficient cells also exhibited increased wound closure in an in vitro scratch assay.ConclusionsThe mouse has three major transcriptional isoforms of the Dlc1 gene that are differentially expressed in various tissues. A mouse with exon 1 of the 6.1 Kb transcript gt resulted in hypomorphic expression of Dlc1 protein and an embryonic lethal phenotype in the homozygous condition, which indicates that this isoform plays a major role in mouse development. The Dlc1 deficient cells showed altered cytoskeleton structure, increased RhoA activity and cellular migration.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.