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Biomedical subjects

F F Becker

Publications and source records attributed to F F Becker.

At least 73 records · Page 4Linked to original sources
PubMed1988

Defective DNA ligase I in Bloom's syndrome cells. Simultaneous analysis using immunoblotting and the ligase-[32P]AMP adduct assay.

Cells from patients with Bloom's syndrome (BS), an autosomal recessive disorder associated with an increased risk of cancer, exhibit genomic instability. Increased numbers of sister-chromatid exchanges (SCE) and delayed DNA chain maturation are typically observed in BS cells. To elucidate the basis for the previously reported decreased DNA ligase I activity in BS cells, simultaneous immunoblot and activity assays for ligase-[32P]AMP adduct formation were performed on extracts from BS and normal lymphoblastoid cell lines. Immunoblot analysis using antibody to DNA ligase I indicate that the amount of the major reactive protein (98 kDa) in normal and BS cells is similar. However, a 50-90% decrease was observed in the ligase activity of the 98-kDa polypeptide in high-SCE BS cells (HG1514 and GM3403c). In contrast, the activity in low-SCE BS cells (HG1554) did not differ significantly from that in normal cells. The data, together with mixing experiments, indicate that the defect in BS ligase I is due at least in part to the loss of ATP binding and/or hydrolytic activity and not to differences in numbers of protein molecules or inhibitory substances. These results suggest that mutation of the DNA ligase I gene may account for the primary metabolic defect in BS.

Adenosine Monophosphate↗
PubMed1988

Analysis of DNA adducts in putative premalignant hepatic nodules and nontarget tissues of rats during 2-acetylaminofluorene carcinogenesis.

Exposure of rats to a standard four-cycle feeding regimen of 0.06% 2-acetylaminofluorene (AAF) results in the formation of putatively premalignant hepatic nodules, but the types and magnitude of DNA adducts formed in these nodules has not been previously examined. By using a sensitive 32P-adduct assay (R. C. Gupta, Cancer Res., 45: 5656-5662, 1985), we analyzed the DNA adduct lesions in individual hepatic nodules at various times during and after exposure to AAF. Kidney, spleen, and testis were included as nontarget tissues. No qualitative difference was observed in the DNA adducts found in hepatic nodules and nontarget tissues; however, quantitative differences occurred. At least one unknown and two known (dG-C8-AF and dG-N2-AAF) DNA adducts were detected, with dG-C8-AF being predominantly (96-98%) formed, in all tissues examined. At the end of the first three weeks of AAF feeding, the concentration of the deacetylated adduct dG-C8-AF in liver (223 fmol/micrograms DNA) was found to be about 2, 6, and 5 times higher than in kidney, spleen, and testis, respectively. The concentration of the N2-acetylated adduct in liver (4.5 fmol/micrograms DNA) was 4-fold higher than in kidney and strikingly higher (51- and 42-fold, respectively) than in spleen and testis. At the end of the fourth feeding cycle, total DNA adducts measured in the hepatic nodules ranged from 30-100 fmol/micrograms DNA, while the "surrounding liver," kidney, spleen, and testis showed 235, 218, 62, and 28 fmol adducts/micrograms DNA, respectively. Sixty days following the cessation of AAF, the binding in both the persistent nodules and liver had decreased to 7% of their respective levels measured at the end of the fourth cycle, while adducts in kidney, spleen, and testis were 32%, 18% and 19%. After 88 days, the binding levels in the nontarget tissues declined further, but no additional adduct removal occurred in the nodules. Our data indicate that (a) although the metabolic apparatus for activation of AAF is diminished in the hepatic nodules, a significant level of adduct formation occurs in the cells of this putative, premalignant lesion, and (b) unlike in the nontarget tissues, repair processes in the premalignant nodules may not be operative several weeks after the cessation of AAF exposure.

2-Acetylaminofluorene↗
PubMed1988

UV-induced photoproducts of 5-methylcytosine in a DNA sequence context.

In order to detect possible m5C photoproducts, highly purified rat liver DNA-cytosine methyltransferase was used to specifically generate m5C with a radioactive methyl group. When these DNAs were subjected to a large dose (10 kJ/m2) of 254 nm or 302 nm ultraviolet light (UVB) to enhance the yield, two labeled photoproducts were detected and isolated by reverse phase HPLC after formic acid hydrolysis. Further studies using acetone as a triplet state sensitizer and UVB irradiation suggested that photoproduct II was activated via a triplet state while the more polar photoproduct I was not. Photoreversion of the purified photoproducts with 10 kJ/m2 254 nm light demonstrated the following reactions: Photoproduct I regenerated m5C, while photoproduct II is split and regenerated m5C and photoproduct I. These results suggest that photoproduct I is monomeric while photoproduct II dimeric, and from the latter's elution position possibly a cyclobutyl type dimer arising from a reaction with an adjacent cytosine. Using d[TTG] and d[Cm5CG] as models of typical sequences, irradiation with 10 kJ/m2 254 nm or 302 nm, respectively, gave rise to a small component having altered mobility in sequencing gels. The altered mobility trinucleotides were resistant to degradation by PI and micrococcal nucleases as expected from photodimerization of the pyrimidine bases. Furthermore, oligonucleotide substrates containing m5C were synthesized and shown to be susceptible to T4 endonuclease v action at locations consistent with d[Cm5C] photodimer formation when irradiated in the UVB range.

5-Methylcytosine↗
PubMed1988

Potassium inhibition of transforming protein P85gag-mos and reversal of the transformed phenotype in 6m2 cells.

K+ at high concentrations (52-72 mM hypertonic KCl) has been reported to induce reverse transformation in the 6m2 cell, which is a clone of normal rat kidney cells (NRK) infected with a temperature-sensitive transformation virus. When exposed to high K+, 6m2 cells grown at the permissive temperature (33 degrees C) exhibit normal morphology and reduced soft agar growth, characteristics of cells grown at nonpermissive temperature (39 degrees C). In the current study, flattening of cells and rearrangement of surface microvilli were demonstrated by scanning electron microscopy to occur within 6 hr of exposure to high K+, similar to the effect of temperature shift to 39 degrees C. Exposure to K+ resulted in a 90% inhibition of P85gag-mos-associated serine kinase activity within 5 min, with a subsequent reduction of up to 75% of the synthesis of this protein. These alterations in the putative transforming protein were similar to those induced by temperature shift and were considered to be the basis for retrotransformation. The cell microtubular system and F-actin cables were affected more slowly by K+ than by a temperature shift to 39 degrees C. The former did not achieve the fine reticulum network seen in NRK cells until 72 hr later, but the latter remained aberrant. The effect on the enzyme might be mediated by alteration in phosphorylation, but the mechanism by which kinase inactivation induces retrotransformation is not yet known.

Actins↗
PubMed1988

Failure of the viable yellow (Avy) and lethal yellow (Ay) genes to enhance chemical induction of thymic lymphomas.

Two dominant mutations in the agouti locus of the mouse, viable yellow, Avy, and lethal yellow, Ay, are associated with increased susceptibility to spontaneous as well as virally or chemically induced tumors. Regardless of the mechanism of initiation, this effect is dependent upon the presence of initiated cells. Although complex, the numerous physiologic alterations associated with these mutations offer an interesting model for the study of endogenous promotion. To date, it has been reported that these genes enhance the growth and early appearance of tumors in all organ sites wherein initiated cells exist, suggesting that the system was pan-promoting. In our current study, however, neither Avy nor Ay enhanced the appearance of chemically induced thymic lymphomas in two mouse strains of differing susceptibility. Thus, the concept of a pan-promoting effect is not valid. However, the presence of Ay did enhance the appearance of chemically induced hepatocellular tumors as has been reported previously for Avy. This striking difference of thymomas from other carcinogenic sequences may make possible a better understanding of the nature of susceptibility to endogenous promoting factors.

Animals↗
PubMed1987

Changes in cell surface charge and transmembrane potential accompanying neoplastic transformation of rat kidney cells.

Free flow electrophoresis measurements have been used to determine the surface charge density of normal rat kidney (NRK) cells and a clone of NRK, designated as 6m2, that exhibit a transformed phenotype at 33 degrees C and a non-transformed phenotype at 39 degrees C. A clone of 6m2, designated 54-5A4, which is transformed at both 33 degrees C and 39 degrees C was also studied. A surface charge density of -1.42 microC/cm2 was obtained for the NRK and non-transformed 6m2 cells at 39 degrees C, whereas at 33 degrees C values of -1.85 and -1.78 microC/cm2 were determined for the transformed 6m2 and 54-5A4 cells, respectively. It was found that 72% of the increased charge that appeared on the transformed 6m2 cells compared with the non-transformed 6m2 cells was RNAase sensitive. The time-dependent decrease in surface charge that accompanied the shift of the 6m2 cells from their transformed to non-transformed state was found to mirror the increase in transmembrane potential previously reported using a fluorescent dye technique, and was also comparable to the reported temporal changes in their morphology and virally-coded protein content.

Animals↗
PubMed1987

Heme enzyme patterns in rat liver nodules and tumors.

Chemically induced rat hepatocyte nodules and carcinomas have a reduced capacity to oxidize drugs. The reduction in monoxygenase activity results largely from the partial loss of cytochrome P-450, a heme-containing terminal electron acceptor. To determine whether the cytochrome P-450 deficit was indicative of an altered heme metabolism, we quantitated four heme-containing proteins in normal rat liver and in rat liver nodules and cancers induced by 2-acetylaminofluorene or diethyl-nitrosamine: cytochrome P-450; cytochrome bs; catalase (EC 1.11.1.6); and tryptophan 2,3-dioxygenase (EC 1.13.11.11). The amounts of these components in nodules were 45%, 88%, 50%, and 59% of normal liver, respectively; in 2-acetylaminofluorene-induced cancers, 65%, 74%, 64%, and 65%, respectively; and in diethylnitrosamine-induced cancers, 40%, 69%, 56%, and 52%. delta-Aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in nodules were 95% and 138% of normal liver, respectively, whereas in 2-acetylaminofluorene-induced cancers, they were 47% and 233%, and in diethylnitrosamine-induced cancers, they were 50% and 175%. These data indicate that four nonmitochondrial liver hemoproteins were diminished to about the same extent in hepatic nodules and cancers. Nodules and cancers also demonstrated an increased capacity for heme degradation, while cancers also demonstrated a decreased capacity for heme synthesis. Thus, the resistance of nodules and tumors to P-450-activated cytotoxic agents may ultimately result from a disturbance in heme metabolism.

2-Acetylaminofluorene↗
PubMed1986

Protective role of thiols in carcinogen-induced DNA damage in rat liver.

Biological thiols are known to play an important role in the detoxification of xenobiotics, including chemical carcinogens. To determine the influence of cellular thiols on carcinogen induction of hepatic DNA damage in the rat, diethylmaleate (DEM) administration was used to deplete intracellular glutathione (GSH). The effects of administration of the synthetic thiols, N-acetylcysteine (NAC) and alpha-mercaptopropionylglycine (alpha MPG), on the induction of DNA lesions were also examined. Pretreatment with DEM reduced liver GSH levels by greater than 70%. As assessed by the technique of alkaline elution, subsequent administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in DNA damage 4 h post MNNG treatment which was 4- to 8-fold greater than that induced in the livers of rats treated with MNNG alone. However, DEM pretreatment had little effect on the extent of DNA damage induced by methylnitrosourea (MNU). DEM alone did not cause any measurable DNA damage. Pretreatment with alpha MPG or NAC reduced MNNG-induced DNA damage by as much as 77%. In contrast, MNU-induced DNA damage was increased by alpha MPG treatment whereas NAC treatment was without effect. These results indicated that in the rat liver, the activity of some DNA alkylating agents may be modulated in varying degree by the concentration of intracellular thiols. These data support the notion that thiols play an important role in protection against carcinogen damage, and that synthetic thiols such as alpha MPG and NAC may be useful as anti-carcinogenic agents against certain carcinogens.

Acetylcysteine↗
PubMed1986

Progression of tumor histiotype during mouse hepatocarcinogenesis associated with the viable yellow (Avy) gene.

Increasing attention has been focused recently upon those factors in carcinogenesis that are responsible for the proliferation of initiated cells and the increasingly aberrant phenotype that they progressively manifest. The agouti locus allele Avy (viable yellow) has been shown to be associated with conditions which favor promotion of cells that have been initiated by a wide variety of causes, in many organs, but has not been previously associated with tumor progression in those systems. In the current study, the presence of the Avy gene in a strain of mice not normally predisposed to hepatocarcinogenesis, C57BL/6N was, for the first time, associated not only with much earlier appearance, but with progression of the histiotype of hepatic tumors, following neonatal administration of diethylnitrosamine. At 52 weeks, 28 C57BL/6N mice demonstrated 7 mouse liver tumors 0.5 cm or greater in diameter, all of more benign histiotype, without associated metastasis. The 31 C57BL/6N-Avy demonstrated 194 mouse liver tumors at that time, 22% of which were of malignant histiotype, 19% of which were associated with metastasis. This system would appear to offer the possibility of identifying the underlying mechanisms for components of the carcinogenic process. In addition, the C57BL/6N-Avy mouse appears to offer advantages as a test animal in bioassay procedures that use the liver as a target organ. Thus, it represents a mouse with little or no spontaneous predisposition to hepatocarcinogenesis, with a predicted short lag period toward response to hepatocarcinogens.

Animals↗
PubMed1986

Heme enzyme patterns in genetically and chemically induced mouse liver tumors.

Chemically induced rat hepatocyte nodules and hepatomas have repeatedly been shown to be deficient in Phase I drug-metabolizing enzymes. Some of these reduced activities are attributable to a diminution of the heme-containing terminal electron acceptor, cytochrome P-450. We recently demonstrated that spontaneous mouse liver tumors exhibit the same deficiency. Therefore, chemically induced and spontaneous liver tumors share common metabolic alterations which are likely to represent intrinsic characteristics of the tumorigenic process and are independent of its etiology. To determine whether the cytochrome P-450 deficit was the result of an altered heme metabolism, we quantitated four heme-containing proteins in normal mouse liver, spontaneous mouse liver tumors, and those induced by a single injection of diethylnitrosamine: cytochrome P-450; cytochrome b5; tryptophan 2,3-dioxygenase (EC 1.13.11.11); and catalase (EC 1.11.1.6). The amounts of these components in spontaneous tumors relative to normal liver were 0.35, 0.68, 0.76, and 0.51, respectively. Similar values were obtained with chemically induced tumors. The enzymes delta-aminolevulinic acid synthase (EC 2.3.1.37), the rate-limiting enzyme in the heme synthetic pathway, and heme oxygenase (EC 1.14.99.3), a degradative enzyme, were also quantitated. The amounts of these enzymes in spontaneous tumor relative to liver were 0.49 and 1.51, respectively. Again, similar values were observed for the chemically induced tumors. Alteration of the latter two enzyme activities may be sufficient for the altered hemoprotein patterns seen in mouse liver tumors. Further, this pattern of metabolic alteration is common to both chemically induced and spontaneous tumors. Thus, tumor resistance to cytotoxic agents activated by the monooxygenase system is not necessarily induced by exposure to these agents, nor as a result of selection.

5-Aminolevulinate Synthetase↗
PubMed1986

Xenobiotic metabolizing enzymes in genetically and chemically initiated mouse liver tumors.

Chemically induced rat liver nodules and cancers characteristically demonstrate a limited capacity to activate xenobiotics to reactive species mainly because of decreased amounts of cytochrome P-450. These lesions also show enhancement of xenobiotic detoxication by such mechanisms as enzymic conjugation or reduction of cytotoxic species. We recently demonstrated a similar pattern of metabolic alteration in spontaneous mouse liver tumors. These findings suggested that certain phenotypic alterations attributed to chronic chemical exposure are inherent in the genetic program for carcinogenesis, and that they may arise independently of chronic exposure. To extend that study, we examined spontaneous and diethylnitrosamine-induced mouse liver tumors for nine enzyme activities commonly reported to be altered in chemically induced rat liver nodules and cancers. The activities of benzo(a)pyrene monooxygenase (EC 1.14.14.1), aminopyrene demethylase, cytochrome P-450 reductase, epoxide hydrolase (EC 3.3.2.3), and UDPglucuronosyl transferase (EC 2.4.1.17) in microsomes from spontaneous tumors relative to those from normal liver were 0.25, 0.43, 1.27, 0.90, and 0.51, respectively. Similar values were obtained with microsomes from chemically induced tumors. The activities of DT-diaphorase (EC 1.6.99.2), glutathione reductase (EC 1.6.4.2), glutathione S-transferase (EC 2.5.1.18), and glutathione peroxidase (EC 1.11.1.9) in cytosol from spontaneous tumors relative to cytosol from normal liver were 2.24, 2.0, 2.43, and 0.31, respectively. Similar values were obtained with cytosol from chemically induced tumors. These results demonstrated that a significant portion of the enzymic phenotype observed in chemically induced rat liver nodules and cancers, which may confer resistance to cytotoxic chemicals, is manifest in spontaneous and chemically induced mouse liver tumors. Further, initiated cells that exhibit this phenotype replicated and progressed in the absence of continued chemical selection.

Aminopyrine N-Demethylase↗
PubMed1985

Potassium-induced reverse transformation of cells infected with a temperature-sensitive transformation mutant virus.

High potassium concentrations altered the morphology and the ability to grow in soft agar in 6m2 cells, a clone of rat kidney cells infected with a temperature-sensitive mutant of Moloney sarcoma virus. Approximately 60% of cells exhibited normal morphology in the presence of 94.8 mM potassium in isotonic medium at the temperature permissive for transformation, whereas 100% were normal at 72 mM potassium in hypertonic media. A significant reduction of growth in soft agar was also induced with these conditions. However, the synthesis ratio of virus-specified transforming protein to marker viral protein was not altered. Na+K+-ATPase might play a role in this reverse-transformation process.

Animals↗
PubMed1985

Perturbation of maintenance and de novo DNA methylation in vitro by UVB (280-340 nm)-induced pyrimidine photodimers.

The effect of pyrimidine photodimers on transmethylation reactions catalyzed by a highly purified rat liver DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) that exhibits maintenance and de novo methylation activities was studied in vitro, using the viral substrates M13 mp9 replicative form (RF) DNA and the hemimethylated analog formed from primed synthesis of phage DNA in the presence of 2'-deoxy-5-methylcytidine 5'-triphosphate. These DNAs were irradiated with UVB (280-340 nm) at 900-3600 J/m2 in the presence of the triplet-state sensitizers acetone or 3-dimethylaminopropiophenone. Under these conditions of irradiation, which approximate solar UV, pyrimidine cyclobutane photodimers were introduced without producing any evidence of single-strand breaks or alkali-sensitive sites [i.e., no (6-4)pyrimidine-pyrimidone photoproducts]. This was confirmed by gel analysis, a T4 UV endonuclease nicking assay specific for cyclobutane-type dimers, and HPLC analysis of the photoproducts. The methylation of irradiated templates by DNA methyltransferase was inhibited in an approximately linear fashion as a function of increasing UVB dose. This inhibition was correlated with the number of lethal photoproducts detected by the simultaneous measurement of the surviving fraction of infectious phage DNA. For approximately the same number of pyrimidine cyclobutane photoproducts introduced, de novo methylation activity was approximately 2-fold more sensitive than the maintenance mode of methylation. The ability of these putatively carcinogenic, pyrimidine photoproducts to inhibit DNA methylation suggests a common mechanism of action with several chemical carcinogens that are known to modify bases.

Animals↗
PubMed1985

Alterations in nonhistone chromatin proteins during hepatocarcinogenesis induced by diverse acting carcinogens.

Nonhistone chromosomal proteins (NHCP) from normal, regenerating rat liver, fetal liver, stages during acetylaminofluorene (AAF) and diethylnitrosamine (DEN) induced carcinogenesis, and resultant primary hepatocellular carcinomas (PHC) were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). These studies sought to determine if changes in proteins putatively involved in catalyzing specific gene expression (NHCP) occur during liver cancer development that might be related to the malignant phenotype. NHCP extracted in high salt-urea buffers, analyzed by 2D-PAGE and silver staining were resolved into some 530-560 polypeptides. Increases in number of NHCP amounting to 8.4, 8.6 and 8.8%, respectively, were detected in AAF induced nodules (AAF-NOD), AAF-PHCs and DEN-PHCs when compared to normal chromatins. The majority of the 51 qualitative changes detected reflected cell cycling and/or reexpression of fetal-NHCP. Within the total changes, seven new NHCP were found only in AAF- and DEN-induced PHCs. Further, four NHCP with isoelectric points and relative molecular weights (pI/MW) of 5.62/19.3, 5.96/30.7, 6.25/46.6 and 8.16/53.5 occurring in both AAF- and DEN-PHC also were found in AAF-NOD, a carcinogenesis stage considered to represent premalignant nodules. Reciprocally, three NHCP of pI/mol.wt.: 6.81/34.0, 5.82/43.7 and 8.18/67.0 present in normal liver, disappeared in all carcinogen involved tissues analyzed. These findings indicate that while AAF and DEN exposure results in a number of qualitative NHCP changes specific for the particular carcinogen, a total of only ten changes, seven inductions and three losses, occurred in common during hepatocarcinogenesis induced by these diverse agents. At least four of these NHCP may prove critical inductions during malignant conversion or alternatively might serve as tumor markers since they appear first in a well characterized premalignant stage and persist in resultant tumors.

2-Acetylaminofluorene↗
PubMed1985

Indistinguishable physical and catalytic properties of DNA methyltransferase from normal rat liver and a transplantable rat hepatocellular carcinoma.

DNA methyltransferase (DMase) was purified 700- and 1002-fold from normal rat liver and transplantable hepatocellular carcinoma 252 (THC 252) nuclei, respectively, using a four-step procedure that included chromatography on phosphocellulose, hydroxylapatite, DEAE-Sephacel and gel filtration on AcA 34. The enzymes had identical characteristics: pI = 7.4-7.6; Mr = 280 000 by gel filtration; preference for methylating double-stranded over single-stranded DNA and hemimethylated over unmethylated DNA templates; and apparent km of 10 microM for dinucleotide units in poly(dC-dG) and 0.5 microM for S-adenosylmethionine (SAM). Thermal inactivation profiles and sulfhydryl group alkylation inhibition curves for fraction III produced very similar single-transition curves, suggesting the presence of a single-functional enzyme species that is indistinguishable between normal and tumor tissue. Single-value Michaelis-Menten kinetics were obtained for fraction IV enzymes with respect to the concentration of SAM and dinucleotide units in poly(dC-dG), suggesting the absence of isozymic or multiple forms of DMase in normal and malignant liver tissues.

Animals↗
PubMed1985

DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene.

A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. In activities of the two largest species, DNA ligase Ia (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50 degrees C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases Ia and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.

2-Acetylaminofluorene↗