Fluorouracil induces apoptosis and surface molecule modulation of peripheral blood leukocytes.

Little is known about the direct effect of chemotherapy on normal peripheral blood leukocytes (PBL) or its contribution to leukopenia. We examined 5'-fluorouracil's (5FU) effect on PBL apoptosis and adhesion molecules' expression in a single-drug solid-tumor model. Possible apoptosis mediators were examined. The study included 32 colorectal cancer patients; apoptosis was determined by annexin-V binding and light-scatter morphology before and after drug infusion. CD18, CD11a, CD11b, and CD63 membranal levels were assayed by flow cytometry. Apoptosis was increased post-5FU administration in neutrophils (PMN), monocytes and lymphocytes (P < 0.05). Levels of Fas receptor and activated caspase 3 did not vary indicating that the process was not mediated by caspase 3 in the timeframe studied. Reduced CD63 on monocytes and decreased CD18 expression on PMN and non-apoptotic monocytes were observed (P < or = 0.05). CD11a,b expression did not vary. Decreased CD18 and CD63 levels were demonstrated in apoptotic and non-apoptotic PBL implying a more direct association with the drug itself.

Transfected mouse c-jun can inhibit transformation of primary rat embryo fibroblasts.

The c-jun gene, which encodes a transcriptional regulatory protein, is the cellular homologue of the transforming gene of avian sarcoma virus 17. In an attempt to assess the biological activities of mouse c-jun, we studied the consequences of its overproduction in an in vitro transformation assay. A c-jun expression plasmid failed to cooperate with either ras, myc or mutant p53 in this focus formation assay. On the other hand, it dramatically inhibited the ability of various oncogene combinations to elicit foci upon transfection into primary rat embryo fibroblasts. Deletion plasmids lacking either the transactivating domain or the leucine repeat of c-jun still displayed a pronounced inhibitory activity. On the contrary, a plasmid encoding only the first 187 amino acids of c-jun had no such activity. The data suggests that enhanced c-jun expression may interfere with the induction or proliferation of transformed cells in this system, and that the inhibitory activity resides in the C-terminal half of the molecule.

Wild-type p53 can inhibit oncogene-mediated focus formation.

Mutant forms of the p53 cellular tumor antigen elicit neoplastic transformation in vitro. Recent evidence indicated that loss of normal p53 expression is a frequent event in certain types of tumors, raising the possibility that such loss provides transformed cells with a selective growth advantage. Thus, it was conceivable that the mutants might contribute to transformation by abrogating normal p53 function. We therefore studied the effect of plasmids encoding wild-type (wt) p53 on the ability of primary rat embryo fibroblasts to be transformed by a combination of mutant p53 and ras. It was found that wt p53 plasmids indeed caused a marked reduction in the number of transformed foci. Furthermore, wt p53 plasmids also suppressed the induction of transformed foci by combinations of bona fide oncogenes, such as myc plus ras or adenovirus E1A plus ras. On the other hand, plasmids carrying mutations in the p53 coding region totally failed to inhibit oncogene-mediated focus induction and often even slightly stimulated it. Hence, such mutations completely abolished the activity of wt p53 that is responsible for the "suppressor" effect. The latter fact is of special interest, since similar mutations in p53 are often observed in human and rodent tumors. The inhibitory effect of p53 was most pronounced when early-passage cells were used as targets, whereas established cell lines were less sensitive. These data support the notions that wt p53 expression may be restrictive to neoplastic progression and that p53 inactivation may play a crucial role in tumorigenesis.

Cotransfection of granulosa cells with simian virus 40 and Ha-RAS oncogene generates stable lines capable of induced steroidogenesis.

Cellular and viral oncogenes are usually defined on the basis of their ability to elicit neoplastic transformation. However, oncogene activity has also been implicated in the control of differentiation. We have tested whether transfection of primary cultured granulosa cells with various oncogenes can yield cell lines that maintain their differentiated properties. Primary granulosa cells were prepared from diethylstilbestrol-treated immature female rats and transfected with simian virus 40 (SV40) DNA or with SV40 plus activated human Ha-RAS oncogene. Transfection with SV40 plus Ha-RAS yielded cell lines that lost response to gonadotropins but, after 48 hr of stimulation with isoproterenol, cholera toxin, forskolin, or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), produced progesterone at levels comparable to those of differentiated primary cells. In contrast, cells transformed only by SV40 lost their ability to produce progesterone. Whereas in primary cell cultures progesterone production was already evident after a 3-hr incubation with 1 mM 8-Br-cAMP, in cotransfected cells progesterone production became evident only after 12 hr. All cotransformed cell lines produced SV40 large tumor antigen as well as human RAS p21 protein. The expression of the expected oncogenes in the various cell lines was confirmed by mRNA analysis. These results suggest that the expression of an activated RAS oncogene in granulosa cells can play a role in preserving inducible steroidogenesis.

Meth A fibrosarcoma cells express two transforming mutant p53 species.

Expression plasmids directing the synthesis of various forms of the p53 cellular tumor antigen were compared with respect to their biological activities. All plasmids encoding wild type p53, derived from two different cDNA libraries, had absolutely no detectable activity when assayed for transformation of primary rat embryo fibroblasts in collaboration with Ha-ras. In contrast, p53 variants carrying point mutations in the protein coding region exhibited at least some transforming activity. Most notably, this was true for both types of mutant p53 cDNA clones isolated from Meth A cells. The data indicate that these cells, derived from a chemically-induced tumor, carry two independently mutated p53 alleles, each encoding a transformationally activated protein. This may imply that the mutations in the p53 gene played a role in the development of the Meth A tumor. Finally, cells overexpressing a transfected mutant p53 exhibit a physical complex between this exogenous p53 and its endogenous counterpart, possibly resulting in the stabilization of the latter.

Specific interaction between the p53 cellular tumour antigen and major heat shock proteins.

The protein p53 is capable of participating in neoplastic transformation and can form specific complexes with the large-T antigen of simian virus 40 (SV40). This interaction probably results in the stabilization of p53 (refs 7,8) and may contribute to SV40-mediated transformation. Several non-SV40-transformed cells also exhibit a stabilized p53 which is present in elevated levels. Recently, this stabilization was shown to coincide with the ability to precipitate a polypeptide (p68) of relative molecular mass (Mr) 68,000-70,000 by anti-p53 monoclonal antibodies. We now report that this co-precipitation indeed represents a specific complex between the two proteins; the complex sediments on a sucrose gradient as a relatively broad peak of 10-14S and can be dissociated in vitro. Furthermore, p68 is the HSP70 heat shock protein cognate, found in elevated levels in a p53-overproducing cell line. On heat-shock treatment of such overproducers, p53 also forms a complex with the related highly inducible HSP68.