A prospective genome-wide study of prostate cancer metastases reveals association of wnt pathway activation and increased cell cycle proliferation with primary resistance to abiraterone acetate-prednisone.

Background: Genomic aberrations have been identified in metastatic castration-resistant prostate cancer (mCRPC), but molecular predictors of resistance to abiraterone acetate/prednisone (AA/P) treatment are not known. Patients and methods: In a prospective clinical trial, mCRPC patients underwent whole-exome sequencing (n = 82) and RNA sequencing (n = 75) of metastatic biopsies before initiating AA/P with the objective of identifying genomic alterations associated with resistance to AA/P. Primary resistance was determined at 12 weeks of treatment using criteria for progression that included serum prostate-specific antigen measurement, bone and computerized tomography imaging and symptom assessments. Acquired resistance was determined using the end point of time to treatment change (TTTC), defined as time from enrollment until change in treatment from progressive disease. Associations of genomic and transcriptomic alterations with primary resistance were determined using logistic regression, Fisher's exact test, single and multivariate analyses. Cox regression models were utilized for determining association of genomic and transcriptomic alterations with TTTC. Results: At 12 weeks, 32 patients in the cohort had progressed (nonresponders). Median study follow-up was 32.1 months by which time 58 patients had switched treatments due to progression. Median TTTC was 10.1 months (interquartile range: 4.4-24.1). Genes in the Wnt/ß-catenin pathway were more frequently mutated and negative regulators of Wnt/ß-catenin signaling were more frequently deleted or displayed reduced mRNA expression in nonresponders. Additionally, mRNA expression of cell cycle regulatory genes was increased in nonresponders. In multivariate models, increased cell cycle proliferation scores (≥ 50) were associated with shorter TTTC (hazard ratio = 2.11, 95% confidence interval: 1.17-3.80; P = 0.01). Conclusions: Wnt/ß-catenin pathway activation and increased cell cycle progression scores can serve as molecular markers for predicting resistance to AA/P therapy.

Localization of polysome-bound albumin and serine dehydratase in rat liver cell fractions.

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [(125)I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [(125)I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [(125)I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5-6 times those released from free polysomes. Anti-rat serum albumin-[(125)I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [(125)I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[(125)I]Fab to free polysomes was also obtained. The [(125)I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[(125)I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.

Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.

Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed.

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles.

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.

Direct association of messenger RNA labeled in the presence of fluoroorotate with membranes of the endoplasmic reticulum in rat liver.

Liver rough endoplasmic reticulum (RER) membranes were isolated from rats given [3H]orotic acid for 48 h (ribosomal RNA [rRNA] label) or for 3 h along with 5-fluoroorotate; this latter procedure permits the labeling of cytoplasmic messenger RNAs (mRNAs) in the absence of rRNA labeling. More than 50% of the labeled mRNA remained attached to membranes of the RER after complete removal of ribosomes with a buffer of high ionic strength in the presence of puromycin. Under similar conditions, membranes retained 40% of their polyadenylate as determined by a [3H]-polyuridylate hybridization assay. Treatment of mRNA-labeled endoplasmic reticulum membranes with pancreatic RNase indicates that the polyadenylate and possibly nonpolyadenylate-pyrimidine portions of the messenger are involved in the binding of mRNA to the membranes. The implication of these results in furthering our understanding of the mechanisms of the translational regulation of genetic expression is discussed.

Cyclic adenosine 3',5'-monophosphate during glucose repression in the rat liver.

Intragastric administration of glucose inhibits the induction of serine dehydratase and tyrosine aminotransferase by glucagon in rat liver, but has no effect on the increase in hepatic adenosine 3',5'-monophosphate resulting from administration of glucagon. Thus, glucose repression in mammalian liver, unlike catabolite repression in microorganisms, appears to operate independently of the amounts of cyclic nucleotide in the cells.

A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.

In a pedigree derived from a mouse treated with the mutagen ethylnitrosourea, a mutation has been identified that predisposes to spontaneous intestinal cancer. The mutant gene was found to be dominantly expressed and fully penetrant. Affected mice developed multiple adenomas throughout the entire intestinal tract at an early age.

Cytochrome P-450 induction by phenobarbital and 3-methylcholanthrene in primary cultures of hepatocytes.

The characteristic hepatocellular changes resulting from phenobarbital administration in vivo, namely an increase in the levels of cytochrome P-450 and proliferation of membranes of the smooth endoplasmic reticulum, have been demonstrated in primary cultures of nonreplicating hepatocytes on floating collagen membranes. Addition of methylcholanthrene to the medium resulted in an increase in cytochrome P-448 within 48 hours, whereas the phenobarbital induction of P-450 required 5 days. These results demonstrate that responses induced in adult liver cells in vivo by phenobarbital can be reporoduced in cultured hepatocytes, contrary to previous reports.

Protein degradation in normal and beige (Chediak-Higashi) mice,.

The beige mouse, C57BL/6 (bg/bg), is an animal model for the Chediak-Higashi syndrome in man, a disease characterized morphologically by giant lysosomes in most cell types. Half-lives for the turnover of [(14)C]bicarbonate-labeled total soluble liver protein were determined in normal and beige mice. No significant differences were observed between the normal and mutant strain for both rapidly and slowly turning-over classes of proteins. Glucagon treatment during the time-course of protein degradation had similar effects on both normal and mutant strains and led to the conclusion that the rate of turnover of endogenous intracellular protein in the beige mouse liver does not differ from normal. The rates of uptake and degradation of an exogenous protein were determined in normal and beige mice by intravenously injecting (125)I-bovine serum albumin and following, in peripheral blood, the loss with time of phosphotungstic acid-insoluble bovine serum albumin and the parallel appearance of phosphotungstic acid-soluble (degraded) material. No significant differences were observed between beige and normal mice in the uptake by liver lysosomes of (125)I-bovine serum albumin (t((1/2)) = 3.9 and 2.8 h, respectively). However, it was found that lysosomes from livers of beige mice released phosphotungstic acid-soluble radioactivity at a rate significantly slower than normal (t((1/2)) = 6.8 and 3.1 h, respectively). This defect in beige mice could be corrected by chronic administration of carbamyl choline (t((1/2)) = 3.5 h), a cholinergic agonist which raises intracellular cyclic GMP levels. However, no significant differences between normal and beige mice were observed either in the ability of soluble extracts of liver and kidney to bind [(3)H]cyclic GMP in vitro or in the basal levels of cyclic AMP in both tissues. The relevance of these observations to the presumed biochemical defect underlying the Chediak-Higashi syndrome is discussed.

Activities of benzphetamine N-demethylase and aryl hydrocarbon hydroxylase in cells isolated from gamma-glutamyl transpeptidase-positive foci and surrounding liver.

The levels of two cytochrome P-450-linked enzymes of xenobiotic metabolism, benzphetamine N-demethylase and aryl hydrocarbon hydroxylase, were determined in cells isolated from gamma-glutamyl transpeptidase (GGT)-positive foci and in cells from surrounding liver obtained from carcinogen-treated inbred F344 rats. Rat liver foci were initiated with diethylnitrosamine (CAS: 55-18-5) and promoted with sodium phenobarbital [(PB) CAS: 64038-21-7] for 4 1/2 or 12 months. The levels of both enzymes were relatively low in the GGT-positive hepatocytes, while the GGT-negative hepatocytes from the surrounding liver had elevated levels of both enzymes comparable to levels seen in rats treated with PB alone. After 12 months of promotion the PB was removed from the diet and the activities of both enzymes fell below the control levels in the GGT-positive hepatocytes and returned to the control levels in the surrounding GGT-negative hepatocytes. Therefore, the cells in the GGT-positive foci contained low levels of these two cytochrome P-450 enzymes in relation to the levels in GGT-negative cells. These levels were responsive to phenobarbital induction, although the induced levels in the GGT-positive cells were much lower than the induced levels in GGT-negative hepatocytes. The liver surrounding the foci responded to phenobarbital induction to the same degree as did the liver of noninitiated rats.

Application of quantitative stereology to the evaluation of phenotypically heterogeneous enzyme-altered foci in the rat liver.

Quantitative stereologic relationships are applied in this report to the evaluation of F344 rat liver foci where the tissue sections exhibit congruent enzyme-altered areas of the several different phenotypes as well as enzyme-altered areas within a larger area of another enzyme alteration, that is, a "focus within a focus.' Quantitation of both the numbers and volume occupied by each of the phenotypes of the enzyme-altered foci was accomplished by the unique logic described in this report. The application of this logic to four representative experimental protocols with the use of three phenotypic markers demonstrated all possible congruent phenotypes as well as a small number of "foci within foci.' The variance of the quantitation of the experimental data was shown to depend on the number of focal transections identified in the sections, the number of sections examined, and the distribution of phenotypic alterations among foci.

Hepatocarcinogenicity of estragole (1-allyl-4-methoxybenzene) and 1'-hydroxyestragole in the mouse and mutagenicity of 1'-acetoxyestragole in bacteria.

Approximately 20% of a dose of estragole, a naturally occurring flavoring agent, was excreted in the urine of outbred male CD -1 mice as a conjuage (presumably the glucuronide) of 1'-hydroxyestragole, Estragole and its 1'-hydroxy metabolite caused significant increases in the incidences of hepatocellular carcinomas in male CD-1 mice that received the compounds by sc injection at 1-22 days of age. Estragole induced hepatocellular carcinomas by 15 months in 23 and 39% of the mice that received total doses of 4.4 and 5.2 mumoles, respectively, and lived to an age of 12 months or more. Of the 12-month survivors given a total dose of 4.4 mumoles of 1'-hydroxyestragole, 70% developed hepatocellular carcinomas; the incidence in mice that received only the vehicle (trioctanoin) was 12%. Multiple tumors ocurred in 5, 28, 64, and 0%, respectively, of the mice in each of these 4 groups. Of the mice given a total dose of 4.4 mumoles of 1'-hydroxysafrole, 59developed hepatocellular carcinomas; 39% of the mice bore multiple liver tumors. As previsously demonstrated for 1'-acetoxysafrole, 1'-acetoxyestragole and 1'-acetoxy-1-allyl-4-methoxynaphthalene reacted nonenzymatically with guanosine and inosine to form adducts. These electrophilic esters were strongly mutagenic for the Salmonella typhimurium missense mutant TA100. 1'-Acetoxyallybenzene had little or no activity in either of these tests. Attempts to demonstrate liver-mediated mutagenicty for 1'-hydroxysafrole and 1'-hydroxyestragole in the bacterial test system were unsuccessful.

Cholangiocellular carcinomas induced in Syrian golden hamsters administered aflatoxin B1 in large doses.

The hepatocarcinogenicity of aflatoxin B1 (AFB1) was compared in male Syrian golden hamsters and inbred F344 rats. AFB1 was administered by gavage 5 days/week for 6 weeks at doses of 1 and 2 mg/kg body weight/day to rats and hamsters, respectively; rate did not survive beyond 3 weeks with doses of 2 mg/kg/day. After 6 weeks the animals either received no further treatment or were given 0.1% phenobarbital sodium in the drinking water. ATPase-deficient foci of hepatic parenchymal cells, neoplastic nodules, and hepatocellular carcinomas were observed in liver sections from AFB1-treated rats killed at 6, 14, or 23 weeks; they were not seen in sections from AFB1-treated hamsters killed at 6, 14, or 46 weeks. Each of the 50 AFB1-treated rats developed hepatocellular carcinomas by 46 weeks; many also had cholangiocarcinomas and mixed hepatocellular-cholangiocellular carcinomas. Hepatocellular carcinomas were found in only 2 of 49 AFB1-treated hamsters by 78 weeks. At this time cholangiocarcinomas were found in 15 hamsters, and microscopic cholangiomas were seen in all of the livers. Compared to the rat, the hamster was resistant to the hepatotoxic and hepatocellular carcinogenic effects of AFB1.

Variability in the oral bioavailability of all-trans-retinoic acid.

BACKGROUND: Orally administered all-trans-retinoic acid (all-trans-RA) can induce complete remission in a high proportion of patients with acute promyelocytic leukemia. A previous pharmacokinetic study in patients with acute promyelocytic leukemia raised the possibility that the absorption of orally administered all-trans-RA is a saturable process that would have significant clinical impact on dosing strategies. PURPOSE: This study was specifically designed to examine the saturability of all-trans-RA absorption by measuring the effect of doubling the oral dose of all-trans-RA on plasma drug concentration in patients receiving long-term oral therapy. METHODS: Six patients with solid tumors received oral doses of 10-mg gelatin capsules of all-trans-RA. Patients were studied on 2 consecutive days after they received 28 days of all-trans-RA administered as two daily 78-mg/m2 doses. The study assigned the patients to two groups. Three patients took a 156-mg/m2 dose on day 28 and a 78-mg/m2 dose on day 29; the other three patients took the lower dose on day 28 and the double dose on day 29. Blood samples for the determination of all-trans-RA plasma concentration were obtained at 30-minute intervals starting just prior to drug administration and continuing for a total of 7 hours. The plasma concentration of all-trans-RA was measured by high-performance liquid chromatography. RESULTS: Plasma concentrations following an oral dose of all-trans-RA were highly variable, with peak concentrations ranging from 0.07 to 1.2 microM for the 78-mg/m2 dose level. Doubling the dose from 78 to 156 mg/m2 increased plasma concentration in all six patients, but the increase was unpredictable and not related to dose, ranging from less than a 1.2-fold to more than a 10-fold increase. CONCLUSION: The current study does not support the hypothesis that the gastrointestinal absorption of all-trans-RA involves a saturable process but instead suggests that absorption is highly variable among patients. This wide interpatient variability suggests that pharmacokinetic drug monitoring may have an important role in the management of patients receiving all-trans-RA.

Effects of the antiestrogens tamoxifen, toremifene, and ICI 182,780 on endometrial cancer growth.

BACKGROUND: Tamoxifen has been shown to promote the growth of human endometrial tumors implanted in athymic mice, and it has been associated with a twofold to threefold increase in endometrial cancer. Toremifene, a chlorinated derivative of tamoxifen, and ICI 182,780, a pure antiestrogen, are two new antiestrogens being developed for the treatment of breast cancer. The effects of these drugs on endometrial cancer are currently unknown. Our objective was to evaluate the effects of toremifene and ICI 182,780 on the growth of human endometrial cancer in athymic mice. METHODS: Athymic, ovariectomized mice were implanted with human endometrial tumors and treated with estrogen, tamoxifen, or the new antiestrogens. RESULTS: The effects of tamoxifen and toremifene on the growth of either tamoxifen-stimulated or tamoxifen-naive endometrial tumors in athymic mice were not substantially different. ICI 182,780 inhibited the growth of tamoxifen-stimulated endometrial cancer, in both the presence and the absence of estrogen. CONCLUSIONS: Toremifene and tamoxifen produce identical effects in our endometrial cancer models. Therefore, it is possible that toremifene, like tamoxifen, may be associated with an increased incidence of endometrial cancer. In contrast, ICI 182,780 inhibited tamoxifen-stimulated endometrial cancer, both in the presence and in the absence of estrogen, suggesting that this drug may be safe with regard to the endometrium, even if it is used following tamoxifen, and that it may not result in an increased incidence of endometrial cancer. Indeed, it is even possible that ICI 182,780 may prove useful as an adjuvant agent in early stage endometrial cancer.

A comparison of polysomal messenger ribonucleoprotein particles from normal and neoplastic rat liver.

Free polysomes were isolated from normal and regenerating rat liver and from Morris hepatomas 7777, 7800, 5123C and 9618A. Sucrose gradient analysis ruled out the possibility of any significant messenger RNA degradation in these polysome preparations. The ethylenediaminetetraacetate-disrupted polysomes were fractionated on oligodeoxythymidylic acid-cellulose columns. The column-bound polyriboadenylic acid-containing messenger ribonucleoprotein particles were eluted with a formamide buffer, precipitated with ethanol, and subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The messenger RNA-associated proteins from the different tissues were qualitatively similar, but two proteins with molecular weights of 66,000 and 109,000 found as minor proteins in normal liver appeared on gels as major protein bands when hepatoma messenger ribonucleoprotein particles were examined. The 66,000- and 109,000-molecular-weight proteins in these particles from regenerating liver appeared quantitatively similar to those isolated from normal liver.

The regulation of serine dehydratase and glucose-6-phosphatase in hyperplastic nodules of rat liver during diethylnitrosamine and N-2-fluorenylacetamide feeding.

Changes in the levels of serine dehydratase and glucose-6-phosphatase induced by dietary stimuli or starvation in hyperplastic nodules of rat liver during diethylnitrosamine or N-2-fluorenylacetamide feeding were studied by immuno- and enzyme histochemical methods. The study was performed during carcinogenesis through a combined method of enzyme histochemistry and radioautography. Serine dehydratase was observed diffusely in the cytoplasm of the original hepatocytes in the periportal zone and was induced markedly during diethynitrosamine feeding but only slightly during N-2-fluorenylacetamide feeding. The enzyme was deficient and not inducible in hyperplastic nodules during their developing phase. Later during the feeding period, however, there was an elevation of the level of serine dehydratase and its inducibility with time in the majority of the nodules. A good correlation was observed between serine dehydratase and glucose-6-phosphatase in their elevated levels and response to enviornmental stimuli. There was a minor group of hyperplastic nodules in which the deficiencies of these enzymes persisted and enzyme induction was not observed. A greater number of hyperplastic nodules with persistent enzyme deficiency was seen during diethylnitrosamine carcinogenesis. These results provide further information about the changing biological nature of hyperplastic nodules with respect to their metabolic adaptability and enzyme levels during hepatocarcinogenesis.

Transcriptional regulation of c-myc during chemically induced differentiation of HL-60 cultures.

Previous studies have demonstrated both an elevated level of cellular myc-homologous RNA in HL-60 cultures and a decrease in this messenger RNA following the chemically induced differentiation of HL-60 cells. A nuclear transcription system isolated from HL-60 cells was used to investigate whether an alteration in the rate of transcription of the c-myc gene was associated with this decrease in myc RNA. Five days after the addition of either 180 mM dimethyl sulfoxide or 60 nM 12-O-tetradecanoylphorbol-13-acetate to HL-60 cultures, transcription of the c-myc gene was markedly reduced when compared with control cultures. This specific decrease was not accompanied by an alteration in either the bulk rate of transcription of the c-myc copy number. This suggests that the decreased cellular myc RNA levels are due to decreased transcription of the myc protooncogene.

Chromatin structure of the c-myc gene in HL-60 cells during alterations of transcriptional activity.

HL-60 cells have an elevated level of cellular myc RNA due to an amplified c-myc gene. Subsequent to chemically induced differentiation of HL-60 cells, both cellular myc RNA levels (Grosso, L. E., and Pitot, H. C., Biochem. Biophys. Res. Commun., 119: 473, 1984) and myc-specific transcription decrease (Grosso, L. E., and Pitot, H. C., Cancer Res., 45: 847-850, 1985). We have compared the primary DNA structure, DNA methylation, and S1 nuclease sensitivity of the myc protooncogene in HL-60 cells before and after chemically induced differentiation. We find no change in the structure or methylation of the c-myc gene. The protooncogene is hypomethylated at CCGG sequences in the 5' region but is extensively methylated at sites detected by sequences homologous to the 3' exon or 3' flanking sequences. Four S1 nuclease-sensitive sites are detected prior to differentiation. After the induction of either myeloid or monocytic differentiation, three of the S1 nuclease-sensitive sites are present. The presence of the fourth S1 nuclease-sensitive site correlates with the transcriptional activity of this gene.

Growth of carcinogen-altered rat hepatocytes in the liver of syngeneic recipients promoted with phenobarbital.

An improved transplantation system for the study of carcinogen-altered hepatocytes is described. This system, which is based on that reported by Laishes and Farber (Cancer Res., 61: 507-512, 1978), involves the transfer of hepatocytes from male F344 animals to syngeneic adult hosts. Unlike the earlier protocol, the recipient rats were fed phenobarbital (PB) rather than the DNA-reactive agent, 2-acetylaminofluorene. gamma-Glutamyl transpeptidase (GGT)-positive hepatocytes were induced in the donor animals by one of three different hepatocarcinogenic treatment regimens. The recipient rats received 0.05% PB in the diet for 3 wk prior to the cell transfer and were maintained on the PB diet for 2 to 7 mo. Hepatocytes from a male F344 donor rat that had received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and had been maintained on 0.05% PB for 12 mo formed GGT-positive colonies and hepatocellular carcinomas in both male and female recipients. No GGT-positive colonies were formed when 0.05% PB was omitted from the diet of the recipients. A 70% partial hepatectomy of the recipients at the time of cell transfer was also essential for the development of colonies and tumors. The mean volume of the colonies was 5 times larger in female recipients than in males, occupying 38% of the total liver volume in the females. GGT-positive foci arose in recipient livers that had received hepatocytes from either a male F344 donor rat treated according to the Solt and Farber [Nature (Lond.), 263: 701-703, 1976] selection protocol or a male F344 donor rat that received a 70% hepatectomy and 30 mg of diethylnitrosamine per kg and was maintained on 0.05% PB for 5 mo. The recipient animals were treated with PB which the initial experiments showed was essential for the development of foci. The number and volume of the foci in the recipient varied according to the treatment regimen that the donor rat received. This system provides a method for analyzing the growth regulation of altered foci at various stages of neoplastic development during hepatocarcinogenesis in the rat in the absence of DNA-reactive selection agents.