Human fibroblasts were subjected to nutritionally induced GI block, followed by release and subsequent entry into S phase, and exposed to nontoxic concentrations of carcinogens in early S phase. Cell transformation occurred as determined by early morphologic cell alterations, anchorage-independent colony formation, cell invasiveness, and augmentation of Ab 376 human malignancy-specific cell-surface antigenic determinant. Methylazoxymethanol acetate was the most potent transforming agent at doses that were negative in toxicity tests. Benzamide (10 FM intracellular concentration), a specific inhibitor of poly(ADPribose) polymerase, prevented transformation in a cell cycle-specific manner, maximal prevention coinciding with early S phase, also characteristic of maximal susceptibility to transformation. Neither an interference of carcinogen deoxyguanosine nucleoside adduct formation nor a chemical reaction between benzamide and carcinogens was detected. Methylazoxymethanol acetate at transforming but nontoxic dose partially inhibited poly(ADP-ribosyl)ation to about the same extent as benzamide. However, simultaneous exposure of cells to both agents in early S phase, resulting in the prevention of transformation, augmented poly(ADPribosyl)ation above the controls. Enzymatic activities ran parallel with the formation of DNA-associating polymer-nonhistone protein adducts that are assumed to regulate the physiological function of chromatin at the structural level.A significant increase in poly(ADP-ribosyl)ation of nonhistone proteins was found in an early precancerous state of hamster liver (1) and in regenerating rat liver (2), suggesting that cellular regeneration may be a common reason for the accelerated enzymatic rates. Cellular regeneration is known to augment the number of cells in S phase and this has been correlated with the facilitation of neoplastic transformation in organs (3) and in cultured C3H/101/3 CL8 mouse fibroblasts (4). Poly(ADP-ribosyl)ation also exhibits a cell cycle-dependent oscillation, a maximum coinciding with S phase after a release of G1 block (5). These observations tend to suggest an as yet undefined connection between (poly ADP-ribosyl)ation, cell cycle, and neoplastic transformation.Ontogenic development (6), the effect of developmental hormones (7-9), and differentiation (10) also coincide with changes in rates of poly(ADP-ribosyl)ation of mainly nonhistone proteins (11, 12) that are thought to regulate selective gene expression (13). Identification of poly(ADP-ribose) as a unique nucleic acid (14, 15) that is covalently bound to presumably DNA-associated proteins may provide a molecular model of chromatin regulation. Ionic environment-dependent helical polymer chains of poly(ADP-ribose) (15) may act as crosslinking agents between regulatory proteins and, depending on their nature and localization, could alter chromatin conformations, expressed as karyological changes that accompany the cell cycle in normal and malignant cells (16,17). The poly(ADP-ribosyl)ation-dependent var...
Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture.
The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors. The distribution of oncogene mRNA and SCC antigen-positive cells was not uniform throughout the tumor. Isolation of cells from the tumors was accomplished by cell culture, growth in soft agar, and growth in the nude mouse. One nontumorigenic immortalized cell line, SCC-83-01-82, isolated by passage through soft agar, was treated with 50 micrograms/ml of methyl methane sulfonate (MMS). These MMS-converted cells subsequently expressed a tumorigenic phenotype. In situ hybridization of the tumors that developed in nude mice revealed increased c-myc and H-ras mRNA expression. Serial passage of the MMS-converted tumors in vivo was accompanied by consistent enhanced c-myc expression. However, the levels of H-ras and keratin mRNA expression decreased with passage in vitro. Northern blot analysis of c-myc and H-ras mRNA levels from the original SCC cell line showed no change in expression following MMS treatment. The data suggest that SCC-83-01-82 is a premalignant cell line established from a mixed cell population in the tumor mass. It can be converted to a malignant phenotype by treatment with MMS, and the persistence of malignancy is under molecular control other than changes in the level of c-myc and ras gene expression.
Replicating DNA was modified by BPDE I to a greater extent than parental DNA when human fibroblast cells were treated with the carcinogen for 30 min in early S phase. Synchronized cells were exposed to 5-bromodeoxyuridine and treated with non-radioactive BPDE I and [methyl-3H]thymidine in early S phase. The density- and tritium-labeled, replicated DNA was separated from parental DNA in a CsCl gradient. The individual carcinogen-DNA adduct levels in both samples were quantitated by using the 32P-postlabeling method. The total modification of replicated DNA was 1.4-2.4 times greater than parental DNA. This difference was mainly reflected by differences in the main adducts, identified as the 3', [5'-32P]bisphosphates of 7R and 7S-BPDE I-dG. Confirmation of the identity of these two specific carcinogen-DNA adducts was accomplished by co-chromatography on t.l.c. with 3H-labeled 3',5'-bisphosphate adducts. The two 3H- and 32P-labeled adducts were isolated and dephosphorylated. The resultant 3H-labeled deoxyribonucleoside adducts were analyzed on h.p.l.c. and identified by co-chromatography with authentic standards. These results suggest that preferential modification of replicating DNA occurs when human cells are treated with BPDE I in early S phase. The ultimate result of this specific modification is the expression of a transformed phenotype.
Addition of benzamide (BZ) at the onset of S phase inhibited expression of the neoplastic phenotype in human foreskin fibroblasts treated in vitro with (±)-7a,8(3-dihydroxy-9f3,10g3-epoxy-7,8,9,10-tetrahydrobenzo[a] [methyl-3H]thymidine (specific activity, 80 Ci/mmol) were purchased from New England Nuclear. Micrococcal nuclease, pancreatic RNase, proteinase K, and Sarkosyl were purchased from Sigma.Cell Culture and Treatment. Human neonatal foreskin fibroblast (HNF) cells were cultured and serially passaged by established procedures (19). These cells were maintained in 75-cm2 flasks in complete growth medium (CM), consisting of Eagle's minimal essential medium (MEM): 25 mM Hepes buffer (pH 7.2) supplemented with 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 50 ,g of gentamicin per ml, 0.2% sodium bicarbonate, and 10% fetal bovine serum. Cells at PDL-5 (population doubling-5) were blocked in the G1 phase by using a nutrient-deficient medium, released from the block, and treated in early S phase with [G-3H] 2769The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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