Biomedical subjects

W K Kaufmann

Publications and source records attributed to W K Kaufmann.

At least 55 records · Page 3Linked to original sources

PubMed1991

Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells.

L1210 cell lines have been described that are sensitive to most platinum compounds (L1210/0), resistant to ethylenediamine (en)-Pt but sensitive to diaminocyclohexane (dach)-Pt (L1210/DDP), and resistant to dach-Pt but sensitive to en-Pt (L1210/DACH). We have examined the effect of the dach and en carrier ligands on the ability of Pt-DNA adducts to inhibit DNA replication. Alkaline sucrose gradient sedimentation was used to determine the influence of both carrier ligands on the inhibition of replicon initiation and DNA chain elongation. Initiation of replicons was inhibited by Pt-DNA adducts to a greater extent than chain elongation in all three cell lines. Inhibition of replicon initiation was affected by the nature of the platinum carrier ligands only in the L1210/DACH cells in which 7.8-fold more dach-Pt adducts than en-Pt adducts were required to reach 63% inhibition. However, a strong carrier ligand effect was observed on the inhibition of DNA chain elongation in both the L1210/DDP and L1210/DACH cell lines. The L1210/DDP cell line required 4-fold more en-Pt adducts than dach-Pt adducts to inhibit DNA chain elongation by 63%. In the L1210/DACH cell line, 2.7-fold more dach-Pt adducts than en-Pt adducts were required for 63% inhibition. The L1210/0 cell line demonstrated no carrier ligand specificity for inhibition of chain elongation. Significant replicative bypass of Pt-DNA adducts was observed even in the L1210/0 cell line in that greater than 50 Pt-DNA adducts per 100 kb were required for 63% inhibition. The same level of inhibition was reached with 1.25 adducts of benzo[a]pyrene diolepoxide I per 100 kb. These data suggest that L1210 cells are capable of substantial replicative bypass of Pt-DNA adducts. Furthermore, the bypass of Pt-DNA adducts is increased in resistant L1210 cells and is markedly dependent on the nature of the platinum carrier ligand.

Animals↗

PubMed1991

Proliferation of carcinogen-damaged hepatocytes during cell-cycle-dependent initiation of hepatocarcinogenesis in the rat.

Hepatocyte proliferation and damage to DNA were characterized during the initiation phase of carcinogenesis in livers of rats that had received a single administration of the methylating agent methyl(acetoxymethyl)nitrosamine (DMN-OAc). Quiescent non-proliferating hepatocytes in intact livers did not appear to be susceptible to initiation by DMN-OAc, whereas proliferating hepatocytes in the S phase appeared to have greatest risk. To characterize the phenomenology of S-phase-dependent initiation further, the fractions of hepatocytes in the S and M phases of the cell cycle were enumerated at various times after treatment with DMN-OAc. Hepatocytes treated when in G1 experienced a delay of up to 20 h in the onset of S phase and a reduced rate of entry into the S and M cycle phases. Hepatocytes treated when in S phase experienced considerable delay in progression to mitosis due to part to inhibition of DNA replication. Hepatocytes treated when in late S/G2 also demonstrated a delay in progression into mitosis. The levels of 7-methylguanine and O6-methyldeoxyguanosine were quantified in the nuclear DNA of proliferating hepatocytes. The kinetics of removal of these lesions appeared to be first-order (half-life = 24 h). Hepatocyte risk of initiation was modeled by a function which summed over time the product of the fraction of hepatocytes in the S phase and the fraction of residual, unrepaired damage to DNA. For hepatocytes treated when in early G1, the time-weighted frequency of premutagenic DNA damage that was present during DNA replication was estimated to be less than half of that for hepatocytes treated when in early S. The results suggest that cell-cycle-dependent variation in sensitivity to initiation of hepatocarcinogenesis may be, in part, due to efficient removal of potentially carcinogenic lesions from DNA during an extended G1. The apparent high sensitivity of hepatocytes in late S/G2 suggests the contribution of additional factors.

Animals↗

PubMed1991

Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes.

Amsacrine↗

PubMed1990

Defective postreplication repair in xeroderma pigmentosum variant fibroblasts.

Postreplication repair (PRR) was quantified in normal human fibroblasts and in xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo[a]pyrene diol epoxide-I (BPDE-I). PRR may be defined as the elimination of discontinuities in the daughter-strand DNA and the replicative bypass of lesions in the DNA template. Pathways of PRR reduce the number of DNA growing points that are blocked at template lesions and increase the rate of growth of nascent DNA on damaged templates. Rates of DNA synthesis and strand growth were measured in solvent- and carcinogen-treated cells by velocity sedimentation analyses of radiolabeled nascent DNA in alkaline sucrose gradients. Logarithmically growing normal fibroblasts displayed D0 values of 6.3 J/m2 and 0.37 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. Under identical conditions, the XP variant cells exhibited D0 values of 1.5-2.0 J/m2 and 0.27-0.31 microM. Pulse-chase experiments were performed in cells synchronized at the beginning of the S phase. Normal and XP variant cells displayed inhibition of DNA strand growth by UV, with D0 values of 21.6 and 7.0 J/m2, respectively. The D0 values for the inhibition of DNA strand growth by BPDE-I were 0.85 microM for the normal cells and 0.62-0.79 microM for the XP variant cells. The inhibitions of DNA replication by UV and BPDE-I were also analyzed in terms of DNA lesion frequencies. Based on the D0 values for inhibition of DNA replication, we concluded that the XP variant cells express maximally 25-33% of the total PRR activity observed in normal fibroblasts after UV treatment. Conceivably, this deficiency in PRR activity results in the XP variant's increased risk of cancers induced by sunlight, because XP variant cells and normal fibroblasts are equally proficient in excision repair. Both normal and XP variant fibroblasts, however, displayed similar PRR activities in response to BPDE-I treatment.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1990

DNA repair endonuclease activity during synchronous growth of diploid human fibroblasts.

DNA-repair endonuclease activity in response to UV-induced DNA damage was quantified in diploid human fibroblasts after synchronizing cell cultures to selected stages of the cell cycle. Incubation of irradiated cells with aphidicolin, an inhibitor of DNA polymerases alpha and delta, delayed the sealing of repair patches and allowed estimation of rates of strand incision by the repair endonuclease. The apparent Vmax for endonucleolytic incision and Km for substrate utilization were determined by Lineweaver-Burk and Eadie-Hofstee analyses. For cells passing through G1, S or G2, Vmax for reparative incision was, respectively, 7.6, 8.4 and 8.4 breaks/10(10) Da per min, suggesting that there was little variation in incision activity during these cell-cycle phases. The Km values of 2.4-3.1 J/m2 for these cells indicate that the nucleotidyl DNA excision-repair pathway operates with maximal effectiveness after low fluences of UV that are in the shoulder region of survival curves. Fibroblasts in mitosis demonstrated a severe attenuation of reparative incision. Rates of incision were 11% of those seen in G2 cells. Disruption of nuclear structure during mitosis may reduce the effective concentration of endonuclease in the vicinity of damaged chromatin. The extreme condensation of chromatin during mitosis also may restrict the accessibility of reparative endonuclease to sites of DNA damage. Confluence-arrested fibroblasts in G0 expressed endonuclease activity with Vmax of 5.5 breaks/10(10) Da per min and a Km of 5.5 J/m2. The greater condensation of chromatin in quiescent cells may restrict the accessibility of endonuclease to dimers and so explain the elevated Km. When fibroblasts were synchronized by serum-deprivation, little variation in reparative endonuclease activity was discerned as released cells transited from early G1 through late G1 and early S. Proliferating fibroblasts in G1 were shown to express comparatively high numbers of reparative incision events in the absence of aphidicolin which was normally used to inhibit DNA polymerases and hold repair patches open. It was calculated that in G0, S and G2 phase cells, single-strand breaks at sites of repair remained open for 30, 19 and 14 sec, respectively. In G1 phase cells, repair sites remained open for 126 sec. Addition of deoxyribonucleosides to G1 cells reduced this time to 42 sec suggesting that the slower rate of synthesis and ligation of repair patches in G1 was due to a relative deficiency of deoxyribonucleotidyl precursors for DNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)

Aphidicolin↗

PubMed1990

Biochemical characteristics of endonuclease activity within the nucleotidyl DNA excision repair pathway of permeable human fibroblasts.

DNA strand breage in response to damage produced by UV (254 nm) radiation was characterized after permeabilization of diploid normal and xeroderma pigmentosum variant fibroblasts. The breakage reaction required ATP, Mg2+ and sucrose for maximal activity and was inhibited by 150 mM Na+ or K+ and 1 mM N-ethylmaleimide. ATP-dependent strand breakage was saturated at UV fluences of above 10 J/m2 and in the presence of DNA precursors breakage was rapidly followed by DNA polymerase and ligase activities to seal the strand breaks. The biochemical features of strand breakage in irradiated permeable cells suggest an enzymatic process. These results, therefore, provide an indication of the biochemical requirements for the rate-limiting strand incision step within the nucleotidyl DNA excision repair pathway.

Adenosine Triphosphate↗

PubMed1989

Pathways of human cell post-replication repair.

Mutagenesis, clastogenesis, and carcinogenesis, may all be S-phase dependent processes within carcinogen-damaged human cells. Carcinogens have been shown to inhibit replicative DNA synthesis in S phase cells and the mechanisms of inhibition have been identified. It is proposed that the sequelae of carcinogen action (mutations, sister-chromatid exchanges, chromosome aberrations) are the consequence of the production of lesions in the DNA template which interfere with the ability of DNA polymerase to synthesize a complementary strand without error. Mis-instructive lesions in the template give rise to base-substitution mutations in nascent strands as DNA polymerase inserts an incorrect but complementary base. Non-instructive base lesions and sterically interfering bulky adducts in the template inhibit DNA polymerase and cause the growing points of nascent DNA strands to be blocked. This blockage perpetuates discontinuities in daughter strands. These discontinuities are eliminated by a process known as post-replication repair. Blocked growing points may be relieved by un-directed insertion of DNA precursors to span the non-instructive lesions. Transient dislocation of the primer terminus from the damaged template may occur at palindromic or repetitive sequences. Reannealing of the primer terminus beyond the site of damage may allow bypass of blocking lesions with a consequence of deletion or insertion of genetic information. DNA at the site of blocked growing points may be a substrate for other enzymes involved in DNA metabolism. Single-strand gaps in daughter strands may be recognized by Rec A-like proteins which catalyze paranemic invasion of sister duplex strands. Recombination intermediates generated at sites of blocked growing points may be resolved by a pathway that produces either sister-chromatid exchanges or the insertion of a patch of parental template DNA within the daughter strand. Single-strand-specific endonuclease may attack regions of denatured DNA at blocked growing points producing double-strand breaks which appear to be intermediates in the formation of chromatid aberrations. The utilization of each of these pathways of post-replication repair will depend upon the precise structure of the template lesion, the sequence context in which the lesion is embedded in the template strand, and stochastic processes.

Cell Cycle↗

PubMed1988

Phenobarbital-dependent proliferation of putative initiated rat hepatocytes.

The mitogenic effects of phenobarbital (PB) were examined using cultures of putative initiated hepatocytes that proliferate and form colonies under conditions in which normal hepatocytes senesce and die. The frequencies of colony-forming hepatocytes in primary cultures isolated 2 weeks after initiation with methyl(acetoxymethyl)nitrosamine or benzo[a]pyrene-7,8-diol-9,10-epoxide(anti) were in the range of 2-38 per million in the presence of PB. Colony-formation frequencies were 0.1 per million in the absence of PB. Proliferative hepatocyte colonies were not observed in cultures grown in serum-free medium containing PB, epidermal growth factor, nor-epinephrine and insulin. The requirement for PB was characterized further using secondary cultures of hepatocytes that had been isolated from a liver 5 weeks after initiation. The colony-forming efficiency of these hepatocytes was about 10% in the presence of 2 mM PB and less than 0.2% in its absence. Colony formation displayed a linear response to concentrations of PB in the range of 0.5-2 mM and a decline above the optimal 2 mM concentration. Autoradiography was used to determine the percentages of hepatocytes in secondary cultures that synthesized DNA in the presence or absence of PB. By the third day after seeding as single cells, hepatocytes exhibited a labeling index of about 50% and this level of labeling was preserved for up to 2 weeks after seeding. Very few hepatocytes were found to synthesize DNA in the absence of PB and most senesced. A small fraction of the colony-forming hepatocytes continued to proliferate in the absence of PB and formed colonies with a high labeling index. These results suggest that the proliferation of initiated hepatocytes in vivo may be conditional upon the presence of the hepatic tumor promoter, PB.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1988

In vitro complementation of xeroderma pigmentosum.

In vitro complementation is a powerful strategy for isolation and characterization of individual components of multienzyme biochemical pathways. Application of the method to elucidate DNA metabolic pathways in prokaryotes enabled the successful identification of pathways of DNA replication and repair. In practice the technique requires the availability of genetic mutants that display defective operation of a selected biochemical pathway, and an in vitro assay system that allows detection of effective operation of the pathway. Xeroderma pigmentosum is a human disease syndrome characterized by partial or severe deficit in the operations of the nucleotidyl DNA excision repair pathway. This pathway of DNA repair appears to respond to DNA lesions which produce substantial distortion of helical structure, the best characterized of which are the UV radiation-induced pyrimidine dimers. This review summarizes a variety of approaches to analysis of the reparative deficiencies in xeroderma pigmentosum by in vitro complementation.

DNA Repair↗

PubMed1987

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)py rene.

Male F344 rats were treated once with (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo(a)pyre ne (BPDE) at various times after a two-thirds partial hepatectomy. Diet containing 0.05% phenobarbital was fed subsequently to promote the development of hepatocellular neoplasms from initiated cells. This combined treatment caused an apparently continuous emergence of hepatocellular neoplasms between 31 and 60 weeks after initiation. Yields of hepatocellular neoplasms (adenomas and carcinomas) seen at 45 weeks after initiation varied according to the time of treatment with BPDE. Rats treated at 15 h after partial hepatectomy, when maximal fractions of proliferating hepatocytes were entering the S phase, displayed the greatest incidence and yield of hepatocellular neoplasms. Rats treated when hepatocytes were early in the prereplicative G1 phase demonstrated a significantly lower incidence and yield of hepatocellular neoplasms. No neoplasms were seen in livers of rats treated with BPDE without prior partial hepatectomy; this indicates the insensitivity or resistance of nonproliferating G0 hepatocytes to initiation by this chemical. The variation in yields of neoplasms could not be attributed to variation in binding of carcinogen to DNA or to rates of DNA repair. The risk of initiation of hepatocytes appeared to be correlated (r = 0.95) with the fraction of hepatocytes that were entering the S phase at the time of treatment with BPDE. These results reveal a significant cell cycle dependency for initiation of hepatocarcinogenesis by BPDE and show that proliferating hepatocytes are at greatest risk when early in the S phase. Quantitation of islands of hepatocellular alteration suggested a similar pattern of cell cycle-dependent sensitivity, although the cycle-dependent variation in island frequencies (1.4-fold) was less than the variation in yields of neoplasms (5-fold). However, 1300-5000 islands were estimated to appear for every one neoplasm that appeared. When viewed within the context of multistep carcinogenesis, and assuming that islands of altered hepatocytes represent a step in this process, then these results suggest that islands may represent populations of cells at least one step removed from neoplasia.

Animals↗

PubMed1987

Cell cycle-dependent initiation of hepatocarcinogenesis in rats by methyl(acetoxymethyl)nitrosamine.

Hepatocyte sensitivity to initiation of carcinogenesis was studied as a function of the cell cycle phase in which damage was incurred. Hepatocytes were stimulated to proliferate by a two-thirds partial hepatic resection, and their proliferation was synchronized further by postsurgical treatment with hydrocortisone. Groups of male F344 rats were given a single administration of methyl(acetoxymethyl)nitrosamine, a highly reactive methylating agent, at various times after two-thirds partial hepatic resection when hepatocytes were in defined phases of the cell cycle. Beginning 3 wk after the treatment and for 37 wk thereafter, rats were fed a diet containing 0.05% phenobarbital to promote the expression of initiated hepatocytes. At 45 wk after treatment with carcinogen hepatocytic neoplasms were enumerated. The greatest yield of neoplasms (5.4 per liver) was observed in the group treated 16 h after two-thirds partial hepatic resection or at the time when proliferating hepatocytes began to enter the S phase of the cell cycle. The least yield of neoplasms (0.8 per liver) was identified in the group treated with methyl(acetoxymethyl)nitrosamine when hepatocytes were early in G1. In the proliferating hepatocytes sensitivity rose continuously during G1 to a peak at the G1-S border and then fell continuously as hepatocytes traversed S, G2, and M. This pattern of response could not be attributed to variation in hepatic esterase which activates methyl(acetoxymethyl)nitrosamine or to variation in methylation of DNA. The results support a model in which carcinogen-induced genetic alterations, occurring at the time of or soon after damaged cells enter the S phase, represent irreversible events that contribute to the initiation of carcinogenesis.

Animals↗

PubMed1987

Factors influencing the initiation by gamma rays of hepatocarcinogenesis in the rat.

F344 male rats were irradiated once with 6 G of cesium 137 gamma rays at various times after a two-thirds partial hepatectomy (PH) and then fed a diet containing the liver tumor promoter phenobarbital to evoke the expression of initiated hepatocytes. Yields of hepatocellular neoplasms were enumerated at 45 weeks after irradiation. Although proliferating hepatocytes in regenerating livers appeared to have increased risk of initiation by gamma rays, there was no apparent variation in risk among groups that were treated at times when hepatocytes were in different portions of the cell cycle (G1, S, G2/M). Gamma irradiation delayed the onsets of DNA synthesis and mitosis by proliferating hepatocytes by 18-20 h. The rate of rejoining of radiation-induced DNA strand breaks was analyzed in primary cultures of hepatocytes; 98% of the strand breaks were rejoined within 30 min after irradiation. Efficient repair of certain types of radiation-induced damage to DNA before the damaged DNA is replicated should cause a substantial reduction in the probability of induction of base-substitution mutations. Hepatocellular islands and neoplasms that were initiated by gamma rays may be derived from proliferating hepatocytes which incurred other radiation-induced DNA damages such as chromosomal aberrations.

Animals↗

PubMed1987

Reparative strand incision in saponin-permeabilized human fibroblasts.

The damage-directed strand incision step in the nucleotidyl DNA excision-repair pathway (NDERP) was characterized in quiescent monolayer cultures of human fibroblasts in which the plasma membrane was selectively permeabilized with saponin. When permeable normal human fibroblasts (NHF) were incubated in a DNA-repair assay mixture lacking the deoxyribonucleoside triphosphate precursors, the numbers of UV-dependent DNA-strand breaks were increased by about 9-fold consistent with the uncoupling of incision from gap-filling DNA synthesis and ligation. In uncoupled NHF omission of ATP reduced the numbers of UV-dependent strand breaks by 84% confirming the requirement for ATP for reparative strand incision. Time-course experiments indicated that the maximum rate of strand incision occurred in the first 10 min of incubation of permeable cells and diminished to 16-28% of this rate between 30 and 60 min of incubation. The initial rate of incision in permeable NHF was estimated to be 20% of that seen in intact fibroblasts. Dose-response studies indicated an initial saturation of strand incision activity at fluences between 10 and 25 J/m2. In permeable group A xeroderma pigmentosum fibroblasts (XPA) few UV-dependent incisions were produced after 10-25 J/m2. In the xeroderma pigmentosum variant (XPV) strain that we studied, strand incisions saturated at a plateau level that was about twice that seen in the NHF strain suggesting the preservation of a higher level of incision activity after permeabilization. After fluences above 50 J/m2 additional strand incision was observed in all cell strains reflecting the activity of a damage-dependent endodeoxyribonuclease that is independent of the NDERP. Saponin-treated fibroblasts were also permeable to pancreatic deoxyribonuclease I and the UV-DNA endonuclease from M. luteus indicating that these preparations may be used for in vitro complementation.

Cell Membrane Permeability↗

PubMed1987

Formation of DNA strand breaks in UV-irradiated human fibroblast preparations.

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m2; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m2. Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblast strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m2 indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m2. Moreover, after 25 or 50 J/m2, strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scraping. Permeabilization in situ saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m2, UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.

Cells, Cultured↗

PubMed1986

Quantitative relationship between initiation of hepatocarcinogenesis and induction of altered cell islands.

We have quantified the initiation of hepatocytic neoplasms and the induction of altered cell islands in regenerating livers of rats given a single treatment with one of three carcinogens before or during the peak of DNA synthesis after partial hepatectomy. For up to 20 wk after treating livers during the peak of DNA synthesis with methyl(acetoxymethyl)nitrosamine (DMN-Ac), hepatocytic neoplasms were not seen. Thereafter, in rats fed the liver tumor promoter, phenobarbital, neoplasms emerged continuously so that by 60 wk after initiation, livers held an average of 5.5 neoplasms. Islands of cellular alteration, identified by their abnormal retention of glycogen on fasting, also appeared to emerge continuously between 20 and 60 wk after initiation. By 60 wk, promoted livers contained about 10,000 islands. In DMN-Ac-initiated, phenobarbital-promoted livers, neoplasms and islands maintained a constant numerical relationship over time with about 1,450 islands emerging for every neoplasm that emerged. This ratio of islands to neoplasms differed according to the type of carcinogen used to initiate hepatocarcinogenesis and depending on whether promotion with phenobarbital was included. In livers initiated with DMN-Ac but not promoted with phenobarbital, the ratio of islands to neoplasms was about 7,750:1. In livers initiated by treatment with (+/-)-7 alpha, 8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9, 10-tetrahydrobenzo[alpha]pyrene at the peak of DNA synthesis and then promoted with phenobarbital, the ratio of islands to neoplasms was 7,200:1. In livers exposed to gamma rays at the peak of DNA synthesis in regenerating livers and promoted, no neoplasms were seen in our sample although islands could be enumerated.(ABSTRACT TRUNCATED AT 250 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1986

Effect of benzo[a]pyrene-diol-epoxide-I on growth of nascent DNA in synchronized human fibroblasts.

In logarithmically growing cultures of human fibroblasts, benzo[a]pyrene-diol-epoxide-I (BPDE-I) treatment inhibits both replicon initiation and DNA chain elongation. In this study we have focused on the effect of BPDE-I treatment on the growth of nascent DNA strands in synchronized human fibroblasts. Cells were collected at the beginning of the S phase by replating confluent cultures in the presence of aphidicolin for 24 h. At 1 h after release from aphidicolin, cells were incubated with [3H]thymidine for 10-15 min and treated with BPDE-I for 10 min. Following different periods of incubation with unlabeled thymidine, cells were lysed on top of alkaline sucrose gradients for analysis of the size distribution of nascent DNA and its average molecular weight. After a brief pulse with the radiolabeled precursor, a unimodal distribution of nascent DNA strands was observed in control cells. Upon chase this distribution was displaced to higher average molecular weights at an approximate rate of 1 X 10(6) daltons/min. Treatment with low doses of BPDE-I (less than 0.2 microM) did not decrease this rate. Indeed the nascent DNA in BPDE-I-treated cells appeared to grow faster than in control cells. This higher rate of DNA strand growth may be related to the inhibition of replicon initiation. Treatment with 0.3 microM or 0.7 microM BPDE-I, which are doses that interfere with DNA synthesis in operating replicons in asynchronous cells, also inhibited the growth of nascent DNA strands in synchronized cells by 22 and 64%, respectively. Because of the simultaneous inhibition of replicon initiation, these values are probably underestimations of the total inhibition of DNA strand growth. The use of synchronized cells in this type of study should aid in the elucidation of mechanisms of replication of carcinogen-damaged DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1986

Xeroderma pigmentosum variant and normal fibroblasts show the same response to the inhibition of DNA replication by benzo[a]pyrene-diol-epoxide-I.

Xeroderma pigmentosum (XP) variant cells are characterized by an abnormal pattern of replication of DNA damaged by 254 nm radiation (u.v.). To see whether benzo[a]pyrene-diol-epoxide-I (BPDE-I) elicits the same response, we have compared the effects of u.v. and BPDE-I on DNA replication in XP variant and normal skin fibroblasts. Doses of u.v. that only affected replicon initiation in normal cells, inhibited DNA strand growth in the XP variant. These results were confirmed by measurements of the rate of growth of single-stranded nascent DNA in cells synchronized at the beginning of the S phase. Identical analyses using BPDE-I, however, indicated that the two cell types were equally sensitive to the inhibitions of both replicon initiation and DNA strand growth. These results indicate that the XP variant phenotype cannot be recognized in vitro by the pattern of response of fibroblasts to the inhibition of DNA replication by BPDE-I.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1986

In vitro colonization ability appears soon after initiation of hepatocarcinogenesis in the rat.

Hepatocytes were isolated from rat livers at various intervals after initiating hepatocarcinogenesis by a single administration of methyl(acetoxymethyl)nitrosamine after a partial hepatectomy. Proliferative hepatocyte colonies were regularly observed in primary cultures derived from initiated livers when these cultures were incubated in medium containing the liver tumor promoter, phenobarbital. Fewer colonies generally were observed in initiated cell cultures that were incubated without phenobarbital and in phenobarbital-induced cultures derived from non-initiated control livers. Because in vitro proliferative activity is a characteristic of malignant hepatocytes these results suggest that the feature of in vitro colonization may characterize a population of carcinogen-altered hepatocytes.

Animals↗