Isolation of a complementary DNA encoding the catalytic subunit of protein kinase A and studies on the expression of this sequence in rat hepatomas and regenerating liver.

A complementary DNA (cDNA) clone (B4) encoding the catalytic subunit of a cAMP-dependent protein kinase (PKAc) was isolated from a lambda gt10 rat brain cDNA library, using a synthetic oligonucleotide probe whose sequence was based on the known amino acid sequence of a bovine cardiac PKAc. Sequence analysis of this clone revealed a region of 1002 nucleotides which encodes a protein that is 92% homologous to amino acids 17-350 of the bovine cardiac PKAc protein. This clone lacks coding sequences for amino acids 1-16 of the latter protein. Nevertheless, it provided a useful probe to analyze expression of the related gene in a variety of systems. Northern blot analyses using a 32P-labeled probe prepared from a 0.6-kilobase PstI fragment of clone B4 revealed an abundant 4.6-kilobase band in rat brain RNA and lesser amounts of this 4.6-kilobase RNA in rat heart and liver. A 4.6-kilobase RNA was also detected in RNA samples obtained from mouse fibroblasts. This probe also detected homologous RNA in a variety of nonrodent species. In subsequent experiments, this cDNA was used as a probe to elucidate the role of PKAc in post-surgical hepatic regeneration and diethylnitrosamine-induced hepatomas in the rat. These experiments revealed that, following partial hepatectomy, PKAc mRNA is decreased 3-fold by 12 h, returning to normal by 72 h; hepatomas showed no consistent pattern of change in PKAc mRNA levels as compared to controls. Our results indicate that this cDNA encodes an isoform of PKAc which is distinct from PKAc-alpha isolated by Uhler et al. (Proc. Natl. Acad. Sci. USA, 83: 1300-1304, 1986) but highly homologous to PKAc-beta isolated by Showers and Maurer (J. Biol. Chem., 261: 16288-16291, 1986), that depression of cAMP-dependent protein phosphorylation may be an important mechanism in the regeneration of mature rat liver but is not a consistent alteration in chemically induced hepatoma, and that this cDNA is useful as a probe for the study of the role of PKAc gene expression in growth control, particularly in rodent species.

Human liver serine dehydratase. cDNA cloning and sequence homology with hydroxyamino acid dehydratases from other sources.

Rat liver serine dehydratase cDNA was used to screen a human liver cDNA library in lambda gt11. One positive clone occurred in every 5,000 clones. Fifteen positive clones were plaque purified. The largest cDNA obtained contained an open reading frame of 987 base pairs, and 5' and 3' noncoding regions of 89 and 317 base pairs, respectively. The deduced amino acid sequence, with a calculated Mr of 34,615, was similar to that of rat liver serine dehydratase except for the absence of a segment consisting of 36 amino acid residues. In vitro transcription/translation with the cDNA resulted in the formation of a polypeptide with an Mr of approximately 35,000, which cross-reacted with the anti-rat serine dehydratase antibody. These results suggest that the human serine dehydratase is structurally cognate with the rat enzyme. Moreover, portions of the sequence postulated to be essential for activity in microbial threonine dehydratases are found in the mammalian serine dehydratases, suggesting that hydroxyamino and dehydratases may have originated from a common ancestor.

Chlorpromazine reduces UV-induced squamous cell carcinogenesis in hairless mice and enhances UV-induced DNA damage in cultured cells.

Administration of the photoactivable compound chlorpromazine (CPZ) to SKH-1 hairless mice via their drinking water (CPZ, 0.01%) significantly reduced the rates of accumulation and yields of squamous cell carcinomas induced by long-term repeated exposures of these animals to solar UV radiation. This protective effect of CPZ was partially reversed in mice given a single injection of ethyl nitrosourea at birth. In in vitro studies, the presence of CPZ (0.2 mM) in mammalian cell cultures enhanced the yield of DNA single-strand breaks induced in the cells by exposure to monochromatic UVA radiation at 334 nm. Collectively, the results suggest that CPZ may exert antineoplastic effects against UV-induced skin tumours by the induction of DNA damage.

Effects of glycerol on lung and liver tumor development.

Mice of several strains (A/J, SWR, MaMyJ, BALB/cByJ, 129J, and C57BL/6J) were treated with the carcinogens 3-methylcholanthrene, urethane, and 4-nitroquinoline 1-oxide and then given 1 or 5% glycerol in the drinking water for up to 4 months. Effects of glycerol on lung tumor multiplicity and incidence were evaluated. The effects of glycerol were variable, and in the majority of experiments glycerol failed to enhance tumor development in mouse lung. Analysis of cell kinetics did not show a proliferative response of alveolar or bronchiolar cells to glycerol. In rats, glycerol did not enhance the appearance of putative preneoplastic liver foci, and in C3H mice it did not increase the incidence of spontaneously occurring liver tumors. It is concluded that glycerol does not increase number or incidence of lung tumors in the mouse strains used, whether the animals are pretreated with a carcinogen or not. Glycerol does not affect liver tumor development.

Use of rat liver altered focus models for testing chemicals that have completed two-year carcinogenicity studies.

Partial hepatectomy (PH) and neonatal rat short-term liver focus models were used to examine the effects of selected chemicals that had been previously tested in the National Toxicology Program (NTP) 2-yr carcinogenicity studies. C.I. Solvent Yellow 14, monuron, chlorendic acid, and 4-hydroxyacetanilide were tested for initiating and promoting activity in the PH model. Chlorendic acid, 4,4'-oxydianiline, 1-amino-2,4-dibromoanthraquinone (ADBAQ), and 4-hydroxyacetanilide were similarly tested in a neonatal rat liver focus model. With the exception of 4-hydroxyacetanilide which was not carcinogenic in the NTP studies, all chemicals tested showed clear evidence of hepatocarcinogenicity. While none of the chemicals showed initiating activity in either the PH or neonatal models, promoting activity, as indicated by increased number, size, or volume fraction of histochemically detected hepatic foci of cellular alteration, was evident for all chemicals with previously demonstrated hepatocarcinogenicity. Liver tumor incidence was documented at 14 months in the PH model and at 300 days in the neonatal model. On the basis of the results obtained from these few chemicals, it is suggested that the use of short-term rat liver focus models may represent a reliable means for identifying chemicals with hepatocarcinogenic potential.

A heritable variant of mouse liver ornithine aminotransferase (EC 2.6.1.13) induced by ethylnitrosourea.

A variant of ornithine aminotransferase (OAT, EC 2.6.1.13) has been detected in an offspring of a male mouse treated with ethylnitrosourea. The evidence presented to support the identification of the protein variant (ENU 2) as altered OAT includes (a) a corresponding 50% decrease in the abundance of a protein, located one charge unit basic to the variant, which comigrates on two-dimensional gel patterns with purified mouse liver OAT; (b) the binding of anti-rat-OAT antibody to the variant; (c) the increased abundance of the variant protein in the livers of mice fed a high protein diet (85% casein); and (d) purification of the variant through an OAT purification protocol.

Isolation and nucleotide sequence of the cDNA for rat liver serine dehydratase mRNA and structures of the 5' and 3' flanking regions of the serine dehydratase gene.

Rat serine dehydratase cDNA clones were isolated from a lambda gt11 cDNA library on the basis of their reactivity with monospecific immunoglobulin to the purified enzyme. Using the cDNA insert from a clone that encoded the serine dehydratase subunit as a probe, additional clones were isolated from the same library by plaque hybridization. Nucleotide sequence analysis of the largest clone obtained showed that it has 1444 base pairs with an open reading frame consisting of 1089 base pairs. The deduced amino acid sequence contained sequences of several portions of the serine dehydratase protein, as determined by Edman degradation. Rat liver serine dehydratase mRNA virtually disappeared from livers of rats fed a protein-free diet for 5 days. Several genomic clones were isolated from two libraries. Determinations of the transcription start site and the structure of the 3' flanking region of the gene indicated that the coded mRNA is 1504 nucleotides long. The 5' promoter region contained a variety of sequences similar to several consensus sequences believed to be important for the regulation of specific gene expression.

Comparative developmental and phenotypic properties of altered hepatocyte foci and hepatic tumors in rats.

Previous investigations in this laboratory have provided evidence that histochemically detectable altered hepatocyte foci and hepatic tumors appearing in rats given a single neonatal treatment with a low dose of carcinogen followed by chronic dietary phenobarbital administration are developmentally independent. The present investigation further evaluates developmental relationships among these lesions. Altered hepatocyte foci were divided into two subclasses consisting of foci that were detectable by histochemical as well as by hematoxylin-eosin staining [designated hist(+)/morph(+) foci] and those foci that were detectable solely by histochemical staining [designated hist(+)/morph(-) foci]. The developmental and phenotypic properties of the hist(+)/morph(-) foci, hist(+)/morph(+) foci, and hepatic tumors were compared in rats initiated once neonatally with different doses of diethylnitrosamine and promoted with dietary phenobarbital from weaning. The morph(+) and morph(-) lesion subclasses were distinguishable on the basis of several developmental characteristics. Hist(+)/morph(+) foci were present at low frequency until at least 150 days after initiation. Although the development of hist(+)/morph(-) foci was essentially complete at that point, the rate of appearance of hist(+)/morph(+) increased significantly. The diethylnitrosamine dose response of the hist(+)/morph(+) foci followed the histochemical marker patterns of the tumor lesion class more closely than that of the hist(+)/morph(-) group. The rates of expression of the hist(+)/morph(+) foci increased with the increasing level of histochemical complexity, whereas the rates of expression of the hist(+)/morph(-) foci groups were inversely correlated to their complexity level. Although the average focus size or diameter in the hist(+)/morph(+) groups was greater than that of the hist(+)/morph(-) foci, the focus growth rates of morph(+) and morph(-) subsets matched for histochemical phenotype were comparable. The complexity level and individual marker distribution patterns of the hist(+)/morph(+) focus class were more similar to tumor patterns than to the distribution patterns of the hist(+)/morph(-) lesion class. The results suggest the following. (a) The development of lesion classes with successively greater deviation from normalcy does not occur via lineal progression from less to more deviated forms within a given lesion class. The three lesion classes appear to develop independently, with the developmental characteristics of each lesion class determined at the time of initiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of rat and mouse ornithine aminotransferase with respect to molecular properties and regulation of synthesis.

A comparative study of the synthesis patterns and molecular properties of mouse and rat ornithine aminotransferase (OAT) was conducted. The two enzymes were found to be very similar with respect to catalytic properties, two-dimensional electrophoresis patterns of tryptic digests, amino acid compositions, and antibody cross-reactivity. In vitro translation assays for OAT synthesis on free polysomes isolated from livers at different times of day showed similar circadian fluctuations in OAT synthesis for both species. However, hybridization measurements revealed no circadian changes in the levels of hybridizable OAT mRNA in these livers. These results demonstrate that the circadian cycling of OAT synthesis is regulated at the level of translation in both the rat and the mouse.

Expression of endogenous retrovirus-like sequences and cellular oncogenes during phenobarbital treatment and regeneration in rat liver.

The expression of two cellular oncogenes (c-myc and c-Ha-ras), the epidermal growth factor receptor gene, and two endogenous retrovirus-like sequences (rat leukemia virus and 30S) was examined in control (nonregenerating) rat livers and at various times after partial hepatectomy. One group of rats had been fed phenobarbital (0.05%) for 16 days prior to the partial hepatectomy. The feeding of phenobarbital (0.05%) itself led to a 65% decrease in the level of epidermal growth factor receptor RNA, but no major change in the level of c-myc, H-ras, rat leukemia virus, or 30S RNAs, in the control rat livers. There was a considerable increase (4- to 5-fold) in the level of c-myc transcripts, at 12 and 48 h after partial hepatectomy in the phenobarbital-treated rats, and at 12 and 24 h in the rats on the control diet. By 72 h, the level of c-myc transcripts returned to normal in both groups of rats. A slight increase (about 1.5-fold) in the level of c-H-ras transcripts was seen at 24 h, which returned to normal levels by 168 h, in the regenerating livers of both the phenobarbital-treated and control diet rats. The regenerating livers displayed a marked decrease (3- to 4-fold) in the level of epidermal growth factor receptor RNA in both the phenobarbital and control diet rats. A marked increase (5- to 6-fold) in the level of transcripts homologous to the endogenous rat leukemia virus-like sequence was seen at 24 h in all of the regenerating livers, but there was no significant change in the level of RNAs homologous to 30S. Thus, the proliferation of normal rat liver cells mimics some but not all of the changes in mRNA levels that we have previously described in rat liver tumors.

Circadian manifestations of barbiturate habituation, addiction and withdrawal in the rat.

The thermal acrophase for the circadian oscillation of core temperature in Charles River male rats fed ad libitum and entrained by light (12 hr dim light and 12 hr bright light) (DL 12:12 hr) occurred near the middle of the dim phase on a control diet of 30% protein. Dietary phenobarbital (0.25%) caused an increase in amplitude of the oscillation (from 0.7 degrees to 1.2 degrees C) and a phase-angle difference (psi-advance) between the zeitgeber and the biological oscillation of about 32 degrees, equivalent to an advance in the thermal acrophase of 2.1 hr in the steady-state. Food consumption was monitored continually and was nearly the same in the two groups; however, animals on the control diet ate around the clock, albeit at a greater rate during dim light than during the bright light phase, whereas rats on phenobarbital started to eat earlier and confined their feeding almost exclusively to early dim phase. This pattern of increase in amplitude of the thermal oscillation and of feeding closely resembling programmed feeding, persisted in phenobarbital-treated animals even in the absence of a dim light-bright light (DL) zeitgeber for eight days. Similar behavior was seen in rats entrained by illumination cycles of 17 hr of dim light and 7 hr of bright light, but with this reduced phase ratio for the zeitgeber, few psi-shifts occurred, and these were smaller than those induced in the group receiving 12 hr of dim light and 12 hr of bright light. In each group, introduction of the drug into the diet and, even more noticeably, removal of the drug from the diet, induced transients of circadian dyschronism that persisted for 4-5 days.

Phenotypically selective promotion of diethylnitrosamine-initiated altered hepatocyte foci by dietary phenobarbital or a topically applied coal-derived organic mixture in male and female rats.

Relative frequencies of diethylnitrosamine (DEN)-initiated foci of altered hepatocytes appearing in response to promotion by either dietary phenobarbital or a topically applied coal-derived organic mixture (CDM) were investigated in male and female rats. The focus population was examined for two histochemical markers, elevated gamma-glutamyl transpeptidase [GG(+)] and iron exclusion [FE(-)], giving rise to 3 detectable focus phenotypes, i.e., GG(+) foci, FE(-) foci, and GG(+)/(FE(-) foci. Frequencies of the 3 phenotypes were quantitated through the use of serial frozen sectioning and computer-assisted image analysis. In agreement with our prior observations, cutaneous exposure to CDM or dietary phenobarbital promoted the expression of DEN-initiated foci. However, the current data showed that this promoting effect of CDM occurred only in females and was restricted to foci with the GG(+)/FE(-) phenotype. Dietary phenobarbital, on the other hand, promoted both the GG(+) and GG(+)/FE(-) phenotypes and was effective in both males and females, although a sex-related differential in the promoting efficiency of phenobarbital was also observed. The pronounced heterogeneity in the responses of the 3 focus phenotypes suggests that each phenotype is the consequence of a specific type of genomic alteration with a specific capacity to undergo phenotypic expression in response to a given promoting stimulus.

Expression of retroviral sequences and oncogenes in rat liver tumors induced by diethylnitrosamine.

The expression of three cellular oncogenes (c-myc, c-Ha-ras, and c-delta-raf), the epidermal growth factor receptor gene, and two endogenous retrovirus-like sequences [rat leukemia virus (RaLV) and 30S] was examined in control rat livers and in 16 liver tumors. The tumors were induced in Sprague-Dawley male and female rats by a single i.p. injection of diethylnitrosamine at 1 or 2 days after birth, followed by dietary exposure to phenobarbital beginning at weaning. Increased expression of c-myc was seen in most of the tumors, but there was no consistent increase or decrease in expression of c-Ha-ras or c-delta-raf. It is of interest that a number of the tumor samples showed a decrease in epidermal growth factor receptor RNA. In all of the tumors, including both hepatocellular adenomas and carcinomas, there was a marked increase in expression of the endogenous RaLV sequence, and over 90% of the tumors displayed increased expression of the 30S endogenous retroviral-like sequence. No or a very low level of expression of the RaLV and 30S sequences was found in the control livers. The extent of expression of the RaLV and 30S sequences in individual tumors did not correlate with the extent of expression of c-myc or c-Ha-ras. Although increased expression of certain endogenous retrovirus-related sequences appears to be a common finding during rat liver carcinogenesis, the significance of this finding remains to be determined.

Effects of separate and combined treatments with gamma radiation and diethylnitrosamine in neonatal rats on the induction of altered hepatocyte foci and hepatic tumors.

To characterize the effects of combined treatments with gamma radiation and diethylnitrosamine (DEN) on the induction of histochemically detectable altered hepatocyte foci and hepatic tumors, we assessed the yields of these lesions in the livers of 150-day-old rats that had been treated neonatally with a single dose of gamma radiation (75 rad, whole body) and i.p.-injected DEN (0.15 mumol/g body wt), either separately or in combination. The combined treatments involved the administration of the two stimuli in both possible sequences, with the interval between treatments set at 1 h. The focus population was examined for two histochemical markers (elevated gamma-glutamyl transpeptidase [GGT(+)] and iron exclusion [FE(-)], giving rise to three detectable focus phenotypes, i.e. GGT(+) foci, FE(-) foci, and GGT(+), FE(-) foci. Frequencies of the three phenotypes were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. GGT(+) focus induction was synergistically enhanced by the combined treatment irrespective of the order in which the two stimuli were administered; the remaining two phenotypes did not show such enhancement. The magnitude of the GGT(+) focus response was significantly greater when the treatment sequence was gamma----DEN as opposed to DEN----gamma. Tumor yields in rats receiving combined gamma--DEN treatment were similar to those in rats receiving the DEN alone, irrespective of the gamma--DEN treatment sequence. These results suggest that phenotypically distinguishable lesions, including foci with different histochemical marker patterns and tumors, originate from specific types of damage at different genetic loci and are developmentally independent; and the expression of the GGT(+) marker per se in altered hepatocyte foci is not a reliable index of incipient hepatic neoplasia.

Effects of rat strain, diet composition, and phenobarbital on hepatic gamma-glutamyl transpeptidase histochemistry and on the induction of altered hepatocyte foci and hepatic tumors by diethylnitrosamine.

To extend our ongoing characterization of modulatory influences on hepatic tumorigenesis, we examined effects of rat strain (Sprague-Dawley versus Fischer), diet composition (semipurified diet versus standard nonpurified laboratory chow), and dietary phenobarbital on the production of gamma-glutamyl transpeptidase (GGT)-positive hepatocyte foci and hepatic tumors initiated by diethylnitrosamine. In addition to GGT-positive foci, we observed, under certain conditions, the appearance of extensive hepatic GGT staining not associated with focal lesions. This elevated nonfocal GGT was found in rats of both strains fed the nonpurified rather than the purified diet, but the level of staining was higher in Fischer than in Sprague-Dawley rats. Enhancement of this nonfocal staining by dietary phenobarbital appeared insignificant. By comparison, frequencies of GGT-positive foci were generally higher in rats fed the semipurified rather than the nonpurified diet, and the frequencies of GGT-positive foci were invariably higher in Sprague-Dawley than in Fischer rats. Moreover, dietary phenobarbital generally enhanced focus production. Assessments of focus and tumor yields among these experimental groups showed that differences in focus frequencies did not correspond closely to differences in subsequent tumor formation. These results document the need to consider the influences of diet and rat strain on experimental end points in designing protocols for hepatocarcinogenesis studies, especially those involving GGT histochemistry. The data also raise questions about the mechanistic relevance of GGT induction to hepatocarcinogenesis and support our prior evidence against the putative lineal relationship between foci and tumors.

2-[(Aminopropyl)amino]ethanethiol (WR1065) is anti-neoplastic and anti-mutagenic when given during 60Co gamma-ray irradiation.

We have studied the effect of 2-[(aminopropyl)amino]ethanethiol (WR1065) on the induction of neoplastic transformation using 10T1/2 cells and on mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus using Chinese hamster V79 cells. Here we report the first observations that treatment of 10T1/2 cells with 1 mM WR1065 for a total of 35 min during irradiation with 60Co gamma-rays significantly reduces the incidence of neoplastic transformation while having no effect on cell viability. In a similar experiment with V79 cells in which 4 mM WR1065 was used, we found a significant reduction in mutation frequency at the HGPRT locus and significant protection against cell killing. These results suggest that WR1065 acts to modulate both acute damage and sub-lethal processes that lead to mutation and neoplastic transformation. Beyond the purely mechanistic approach of these studies, the potential application of these agents to minimizing the long-term neoplastic effects of radiation or chemotherapeutic agents currently in use for treating potentially curable cancer patients should be further investigated.

Synergistic induction of altered hepatocyte foci by combined gamma radiation and diethylnitrosamine administered to neonatal rats.

To investigate mechanisms underlying the formation of carcinogen-induced altered hepatocyte foci, we histochemically examined the livers of 150-day-old rats that had been treated neonatally with single doses of gamma radiation (75, 150 or 300 rad, whole body) and i.p.-injected diethylnitrosamine (DEN, 0.15 mumol/g body wt) either separately or in combination. Three focus phenotypes, showing the elevated gamma-glutamyl transpeptidase [GG(+)] and/or the iron-exclusion [Fe(-)] histochemical marker(s) were quantitated through the use of serial frozen sectioning techniques and computer-assisted image analysis. DEN and gamma radiation each induced foci when given separately but total focus yield per cm3 of liver was more than 10-fold greater in DEN-treated than in irradiated rats and approximately 3-fold higher in females than in males. Combining the DEN and radiation treatments synergistically increased total focus yields for both sexes, although this response declined with increasing radiation dosage. For rats receiving DEN alone, the sex-dependent differential in total focus yield was due to the higher frequencies in females of the iron-excluding phenotypes [Fe(-) alone and Fe(-) + GG(+)]; the frequencies of foci showing only GG(+) were similar in both sexes. In contrast, the enhancement in total focus yield resulting from the combined DEN--radiation treatments was primarily a consequence of increases in the foci with GG(+) alone. The results suggest that (a) qualitatively different types of genetic damage (carcinogen-induced point mutations and radiation-induced rearrangements) may interact synergistically in the induction of phenotypically altered cells and (b) separate genetic loci are involved in the sex-mediated modulation of focus production and the synergistic enhancement of focus production by DEN--gamma radiation interactions.

Evidence for growth heterogeneity among foci with different phenotypes in the population of altered hepatocyte foci induced by a single neonatal treatment with carcinogen.

Relationships between phenotypic and growth characteristics of carcinogen-induced altered hepatocyte foci were investigated. Male and female rats were given a single i.p. injection of carcinogen (diethylnitrosamine or benzo[a]pyrene) within 1 day after birth and were exposed to dietary promoter (phenobarbital) beginning at weaning. Groups of these rats were then killed at intervals, and their livers were examined for foci exhibiting various phenotypic markers through the use of serial frozen sectioning techniques, histochemical staining and computer-assisted image analysis. These procedures permitted the identification and sizing of foci with different specific phenotypes (identities of focus markers) within each phenotypic complexity level (number of markers per focus). The data suggest that foci growth rates differ with respect to specific focus phenotypes within complexity levels. This observation complements previous demonstrations of a direct relationship between foci growth rates and levels of phenotypic complexity and indicates that the observed diversity of focus phenotypes reflects true biological diversity within the focus population. Given the prior evidence for (i) the stability of focus phenotypes; (ii) the rapid emergence of phenotypically dissimilar foci following a single carcinogen treatment; and (iii) the production of foci by single initiation events, we suggest that each proliferatively and phenotypically distinct member of the focus population reflects the occurrence of a lesion at a unique genetic locus during initiation.