Modulation of mitogenesis by liver fatty acid binding protein.

Liver fatty acid binding protein (L-FABP), a cytoplasmic 14 kDa protein previously termed Z protein, is conventionally considered to be an intracellular carrier of fatty acids in rat hepatocytes. The following evidence now indicates that L-FABP is also a specific mediator of mitogenesis of rat hepatocytes: a. the synergy between the action of L-FABP and unsaturated fatty acids, especially linoleic acid, in the promotion of cell proliferation; b. the specific requirement for L-FABP in induction of mitogenesis by two classes of nongenotoxic hepatocarcinogenic peroxisome proliferators (amphipathic carboxylates and tetrazole-substituted acetophenones); c. the direct correlation between the binding avidities of different prostaglandins for L-FABP and their relative growth inhibitory activities toward cultured rat hepatocytes; d. the temporal coincidences between the covalent binding to L-FABP by chemically reactive metabolites of the genotoxic carcinogens, 2-acetylaminofluorene and aminoazo dyes, and their growth inhibitions of hepatocytes during liver carcinogenesis in rats; e. and f. the marked elevations of L-FABP in rat liver during mitosis in normal and regenerating hepatocytes, and during the entire cell cycle in the hyperplastic and malignant hepatocytes that are produced by the genotoxic carcinogens, 2-acetylaminofluorene and aminoazo dyes. These actions of L-FABP are consistent with those of a protein involved in regulation of hepatocyte multiplication. Discovery that L-FABP, the target protein of the two types of genotoxic carcinogens, is required for the mitogenesis induced by two classes of nongenotoxic carcinogens points to a common process by which both groups of carcinogens promote hepatocyte multiplication. The implication is that during tumor promotion of liver carcinogenesis, these genotoxic and nongenotoxic carcinogens modify the normal process by which L-FABP, functioning as a specific receptor of unsaturated fatty acids or their metabolites, promotes the multiplication of hepatocytes.

Liver fatty acid-binding protein: specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators.

Peroxisome proliferators (PP) are a diverse group of chemicals that induce dramatic increases in peroxisomes in rodent hepatocytes, followed by hypertrophy, hepatomegaly, alterations in lipid metabolism, mitogenesis, and finally hepatocarcinomas. Termed nongenotoxic carcinogens, they do not interact with DNA, are not mutagenic in bacterial assays, and fail to elicit many of the phenotypes associated with classic genotoxic carcinogens. We report here that the mitogenesis induced by the major PP class, the amphipathic carboxylates, and by the tetrazole-substituted acetophenones specifically requires liver fatty acid-binding protein (L-FABP) in cultured rat hepatoma cells transfected with the sense cDNA of L-FABP, in contrast to L-FABP-nonexpressing cells transfected with its antisense cDNA. The mitogenic actions of L-FABP were protein-specific, inasmuch as no other protein in the nonexpressing cells could act like L-FABP. L-FABP was previously shown not only (i) to interact covalently with metabolites of the two genotoxic carcinogens 2-acetylaminofluorene and aminoazo dyes during liver carcinogenesis, but also (ii) to bind noncovalently the two classes of PP in vitro with avidities that correlate with their abilities to elicit peroxisomal enzymatic responses, and (iii) together with unsaturated fatty acids, especially linoleic acid, to promote multiplication of the transfected hepatoma cells in culture. The convergence of the two types of genotoxic carcinogens with the two classes of PP nongenotoxic carcinogens, and also with unsaturated fatty acids, at L-FABP actions in inducing mitogenesis allows the following hypothesis. During tumor promotion of carcinogenesis in vivo, these groups of genotoxic and nongenotoxic carcinogens act on the normal process by which L-FABP, functioning as a specific receptor of unsaturated fatty acids or their metabolites, promotes hepatocyte proliferation.

Growth promotion of transfected hepatoma cells by liver fatty acid binding protein.

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L-FABP) of rat liver cytosol. In search for the roles of L-FABP in hepatocytes, we previously stably transfected rat L-FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L-FABP RNA or protein, thereby providing a zero-background, homologous cell model of L-FABP-expression suitable for controlled studies of its intracellular functions in hepatocyte-derived cells. The present study demonstrates the abilities of L-FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L-FABP-nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L-FABP-expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5-fold (avg.) as many L-FABP-expressing cells than L-FABP-nonexpressing cells. In addition, the L-FABP-expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L-FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to eliminate the differences in behavior of the two cell types in the serum-free medium. The unsaturated fatty acids, oleic acid (18:1 omega 9), linoleic acid (18:2 omega 6), alpha-linolenic acid (18:3 omega 3), and arachidonic acid (20:4 omega 6), at 1 microM markedly elevated the level of DNA synthesis in the more depressed control L-FABP-nonexpressing cells and moderately raised it in the less depressed L-FABP-expressing cells. In accord, the control L-FABP-nonexpressing cells needed 10(-6)-10(-5) M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 microM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Specific growth stimulation by linoleic acid in hepatoma cell lines transfected with the target protein of a liver carcinogen.

The hepatic carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) was shown previously to interact specifically with its target protein, liver fatty acid binding protein (L-FABP), early during hepatocarcinogenesis in rats. In search of the significance of the interaction, rat L-FABP cDNA in the sense and antisense orientations was transfected into a subline of the rat hepatoma HTC cell line that did not express L-FABP. After the transfections, the basal doubling times of the cells were not significantly different. However, at 10(-5)-10(-7) M, linoleic acid, which is an essential fatty acid, a ligand of L-FABP, and the precursor of many eicosanoids and related lipids, stimulated the incorporation of [3H]thymidine in three randomly isolated and stably transfected cell clones that expressed L-FABP, but virtually did not stimulate the incorporation of [3H]thymidine in three L-FABP-nonexpressing clones transfected with the antisense DNA. Linoleic acid at 10(-6) M increased cell number almost 3-fold (38% vs. 14%; P less than 0.0001) and thymidine incorporation nearly 5-fold (23.2% vs. 4.9%; P less than 0.001) in the L-FABP-expressing cells compared to that in the transfected nonexpressing cells. L-FABP acted specifically and cooperatively with linoleic acid, inasmuch as all the proteins other than L-FABP in the transfected L-FABP nonexpressing cells and four other fatty acids (gamma-linolenic acid, dihomo-gamma-linolenic acid, arachidonic acid, and palmitoleic acid) were unable to effect a significant elevation or difference in the level of DNA synthesis that was attributable to the transfection. Metabolism of the linoleic acid to oxygenated derivatives was apparently necessary, since the cyclooxygenase inhibitor indomethacin partly inhibited and the antioxidant lipoxygenase inhibitors nordihydroguariaretic acid and alpha-tocopherol completely abolished the growth stimulation. The evidence supports the idea that L-FABP, the target protein of the liver carcinogen, acts specifically in concert with oxygenated metabolites of linoleic acid to modulate the growth of hepatocytes.

Preferential binding of growth inhibitory prostaglandins by the target protein of a carcinogen.

Liver fatty acid binding protein (L-FABP) is the principal target protein of the hepatic carcinogen N-(2-fluorenyl)acetamide (2-acetylaminofluorene) in rat liver. In addition, the cyclopentenone prostaglandins (PG), PGA, PGJ2, and delta 12-PGJ2, inhibit the growth of many cell types in vitro. This report describes the preferential binding of the growth inhibitory prostaglandins by L-FABP and the reversible inhibition of thymidine incorporation into DNA by PGA2 and delta 12-PGJ2 in primary cultures of purified rat hepatocytes. As a model ligand, [3H]PGA1 bound to L-FABP specifically, reversibly, rapidly, and with high affinity. Its dissociation constants were 134 nM (high affinity) and 3.6 microM (low affinity). The high-affinity binding of [3H]PGA1 was 9- and approximately 13-fold more avid than the binding of the conventional fatty acid ligands, oleic acid and arachidonic acid, respectively. The abilities of different prostaglandins to compete with the high-affinity binding of [3H]PGA1 correlated with their growth inhibitory activities reported previously and here. The growth inhibitory cyclopentenone prostaglandins (PGA1, PGA2, delta 12-PGJ2, and PGJ2) were the best competitive ligands, intermediate competitors were the weak growth inhibitors PGE1 and PGD2, and the poorest competitors were PGE2 and PGF2 alpha, which stimulate rather than inhibit DNA synthesis in rat hepatocytes in primary culture. The in vitro actions of L-FABP are compatible with those of a specific and dissociable carrier of growth inhibitory prostaglandins in rat hepatocytes and suggest that the carcinogen may usurp the cellular machinery of the growth inhibitory prostaglandins.

Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein.

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.

Normal hepatocytes exhibiting histone H3 with antibody accessible sites that are cryptic in carcinogen-altered hepatocytes.

A Mr 14,000 polypeptide (p14), identified as liver fatty acid binding protein, in normal liver cytosol was shown previously to be the principal target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during hepatic carcinogenesis in rats. Immunohistochemical analyses using rabbit antiserum against pure p14/liver fatty acid binding protein revealed marked increases in the levels of the protein in cytoplasm specifically during mitosis in normal and regenerating hepatocytes, and throughout the cell cycle in hyperplastic and malignant hepatocytes brought about by carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene) or 3'-methyl-4-dimethylaminoazobenzene. Present also in normal hepatocytes was a nuclear antigen that was not detected in the hyperplastic hepatocytes, benign hepatocytic adenomas, and hepatocellular carcinomas produced by these carcinogens. The nuclear antigen was discerned to be a Mr 17,000 polypeptide (p17) in extracts of normal liver nuclei and nucleosomes. In the present study, the p17 was purified by high-performance liquid chromatography and identified as being the three variants of histone H3, based on common molecular size, amino acid composition, electrophoretic migration in Triton-acetic acid-urea gels, and Western blot and histochemical reactions using affinity-purified antibodies. The histone H3 of all tested organs reacted specifically with the antiserum in Western blots following sodium dodecyl sulfate gel electrophoresis. In contrast, in a survey of 23 normal rat organs, nuclei of virtually only hepatocytes were reactive immunohistochemically. In view of the exceptional immunohistochemical reactivity of nuclei of normal hepatocytes, attributable to accessible histone H3, and the lack of such reaction in carcinogen-altered hepatocytes, the collected evidence indicates that normal hepatocytes contain uniquely available histone H3 sites that become cryptic during the chemical carcinogenesis.

Late target protein of the carcinogen N-2-fluorenylacetamide in rat liver.

In previous studies, administration of a radioactive tracer dose of the liver carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), to normal or carcinogen-fed rats led to the presence of one or two principal labeled carcinogen: protein complexes in liver cytosol. The early target Mr 14,000 protein of the carcinogen in normal rats was identified as being liver fatty acid-binding protein and was associated in hepatocytes with normal mitosis and the cell proliferation brought about by either of the two liver carcinogens, FAA or 3'-methyl-4-dimethylaminoazobenzene. Continued ingestion of any of the three hepatocarcinogens, FAA, 3'-methyl-4-dimethylaminoazobenzene, or ethionine, resulted in the progressive loss of the early radioactive complex and the concurrent gain in liver cytosol of the late carcinogen: protein complex (Mr approximately 150,000) formed by the tracer dose of FAA. Present attempts to extract FAA derivatives from the late carcinogen: protein complex with organic solvents indicated that virtually all of the carcinogen was apparently covalently bound to the resultant denatured protein. It is unknown whether the covalent interaction occurred in vivo or as an accompaniment of the protein denaturation associated with the solvent extractions. In support of a possible noncovalent interaction, treatment of the unextracted complex with sodium dodecyl sulfate, urea, and beta-mercaptoethanol followed by electrophoresis readily dissociated the majority of the bound carcinogen. The late carcinogen: protein complex was shown to contain a 55 kDa subunit (p55), which was purified to homogeneity according to molecular size. The subunit is a relatively basic polypeptide with a pI of 8.4 to 8.6. In Western blots using rabbit immunoglobulins against the p55, the late target protein was found to be present at low concentrations in liver cytosols of normal rats, and was induced to relatively high levels by ingestion of the carcinogen for 3 to 5 weeks. The induction of high levels of the late target protein explains in part the progressive elevation in content of the late carcinogen: protein complex in rat liver during carcinogenesis by FAA. The isolated p55 was susceptible to a spontaneous stepwise breakdown, resulting in a ladder of decreasing molecular sizes with an average unit difference of 5.8 kDa per step over six size intervals. The p55 subunit was detected in nonhepatic organs of normal rats, but unlike levels in liver, the levels there were not affected by ingestion of the carcinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens.

Elevated expression and cell cycle deregulation of a mitosis-associated target polypeptide of a carcinogen in hyperplastic and malignant rat hepatocytes.

A cytoplasmic polypeptide with a molecular weight of 14,000 (p14) was previously found to be the principal covalent target protein of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene), early during carcinogenesis in rat liver. The level of immunodetected p14 was markedly increased specifically during all stages of mitosis of hepatocytes in normal and regenerating partially hepatectomized livers. In addition, the polypeptide appeared to be immunologically and behaviorally related to a polypeptide with a molecular weight of 17,500 that is tightly bound to nucleosomes of chromatin in hepatocytes. This report describes the actions of the two polypeptides during hepatocarcinogenesis induced by ingestion of 3'-methyl-4-dimethylaminoazobenzene or N-2-fluorenylacetamide. Both carcinogens acted similarly in bringing about characteristic responses of the two polypeptides, detected immunohistochemically with specific rabbit antiserum and peroxidase-antiperoxidase complex. Four types of discrete hepatocytic lesions were observed. The earliest and least aberrant were hyperplastic foci of proliferating hepatocytes, which generally displayed markedly higher levels of immunostained p14 in cytoplasm, compared to levels in normal diploid hepatocytes. Furthermore, the very high concentrations of p14 were continuously present during cell interphase, in contrast to those in normal and regenerating hepatocytes, in which the elevation is restricted to the period of mitosis. Later-arising lesions were acidophilic adenomas of hepatocytes, which were characterized by quiescent morphology, fairly uniform and bulky eosinophilic cytoplasm, high levels of glycogen, and small delicate nuclei. These cells usually displayed little cytoplasmic p14. Coexistent hepatocytic lesions, i.e., mixed basophilic adenomas, exhibited considerable morphological heterogeneity, often slightly basophilic cytoplasm, and variable immunostain of cytoplasmic p14 during interphase. The fourth lesions were hepatocellular carcinomas, which continuously demonstrated much higher than normal levels of cytoplasmic p14 during cell interphase. During mitosis in the four types of hepatocytic lesions, the levels of immunostained p14 were usually further elevated above those in interphase, regardless of whether they were already very high during interphase in hyperplasia and malignancy, or low in acidophilic adenomas. All four kinds of carcinogen-altered lesions usually displayed little of the detectable Mr 17,500 polypeptide in nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

A new nucleosomal protein in normal liver related to the cytoplasmic polypeptide target of a carcinogen.

Normal adult rat liver contains a nucleosomal protein that is related to the principal target polypeptide of a carcinogen in cytoplasm. Normal rat liver was found previously to contain a 14 000-dalton polypeptide that is the principal cytosolic target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), early during hepatocarcinogenesis. Elevated levels of immunohistochemically detectable target polypeptide in cytoplasm are associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes. A putatively related 17 500-dalton polypeptide was shown to be tightly bound to chromatin of normal liver nuclei. We report here that purified nucleosomes from normal rat liver contain the bound 17 500-dalton protein. Nuclei were digested with micrococcal nuclease, and the resultant nucleosomes were resolved into size classes by density gradient sedimentation. The monomers, dimers, and trimers of nucleosomes possessed bound 17 500-dalton polypeptide, as determined by SDS gel electrophoresis followed by immunoelectroblot analyses. Alterations in the levels of the two polypeptides were shown previously to occur during liver carcinogenesis by FAA and 3'-methyl-4-dimethylaminoazobenzene. The findings support the possibility that the 17 500-dalton polypeptide may function normally in a role related to the replication or expression of the hepatic genome, and may be connected with changes in hepatic genic activity brought about by the carcinogens.

Induction of epidermoid differentiation by cyclic adenine nucleotide in cultured mammary tumors of mice.

In search of the degree of responsiveness of mammary adenocarcinomas to signals of differentiation, mouse mammary tumors were induced to undergo a course of development leading to multiple foci of squamous metaplasia, and subsequently a differentiation manifested by marked keratinization. The mammary tumors had spontaneously arisen in the preneoplastic mammary outgrowths of the transplantable lines, D1, MH5, and MH9, after their long-term implantation in gland-free mammary fat pads of virgin BALB/c mice. The inductions were produced in cultured fragments of mammary tumors by incubation for 9 days in the cyclic adenine nucleotide, N6-O2'-dibutyryl cyclic AMP, at 0.1 mM, without or with prostaglandins E1, E2, and B1, each at 5 micrograms/ml, and 1 microM papaverine. The N6-O2'-dibutyryl cyclic AMP alone was as active in the mammary tumors derived from the D1 and MH9 preneoplastic outgrowths as was the entire mixture of inducers. Intracellular cyclic adenine nucleotide may presumably be the specific mediator of the inductive process, presumably being elevated synergistically by entry of the N6-O2'-dibutyryl cyclic AMP, by induction of adenyl cyclase by prostaglandin E1 and E2, and through inhibition of phosphodiesterases by papaverine. Epidermidalization occurred to equal extent in well-differentiated and anaplastic mammary adenocarcinomas, indicative that mammary tumor progression did not affect the susceptibility to this course of development and differentiation. Mammary gland epithelium retains its susceptibility to multifocal epidermidalization in organ culture throughout the gradient of neoplastic transformation and progression toward decreasing growth regulation, starting from normal mammary gland, next preneoplasia (both reported previously), then well-differentiated neoplasia, and lastly anaplastic cancer. The findings support the existence of a common or closely associated pool of progenitor cells for the alveolar and epidermoid courses of development and differentiation in mammary gland. Induction of squamous metaplasia and abundant keratinization in both the well-differentiated and anaplastic mammary adenocarcinomas caused some of the cells to differentiate terminally and to die.

Mitosis in hepatocytes is generally associated with elevated levels of the target polypeptide of a liver carcinogen.

Rat hepatocytes have previously been found to contain in their cytoplasm a 14,000-dalton polypeptide that: is markedly and specifically increased in concentration during the infrequent mitoses that occur in hepatocytes of adult normal liver; is apparently related to a 17,500-dalton polypeptide that is tightly bound to the chromatin of nuclei of normal adult liver; is the principal covalent target of the carcinogen, N-2-fluorenylacetamide (FAA; 2-acetylaminofluorene), early during liver carcinogenesis; is present at highly elevated levels in the proliferating hepatocytes of hyperplastic foci brought about by the two liver carcinogens, FAA and 3'-methyl-4-dimethylaminoazobenzene; and is present at a greatly depressed level in the mitotically nonresponsive parenchyma that surrounds these hyperplastic foci. In the present investigation, we examined the levels of the two polypeptides in hepatocytes undergoing cell division at different rates in livers of diverse normal and regenerative states. Using immunohistochemical techniques, both polypeptides were detected in developing hepatocytes in as early as 15- and 19-day rat fetuses. With the increasing maturity of fetal and neonatal livers in normal rats, a greater percentage of dividing hepatocytes exhibited a higher concentration of the 14,000-dalton target polypeptide than that seen in adjacent interphase hepatocytes. The percentage of mitotic hepatocytes with an elevated level of the polypeptide increased progressively with hepatic development as follows: 38% in 15-day fetuses, 50% in 19-day fetuses, 85% in 1-day neonates, between 79% and 93% until 28 days of age, and finally, 99% in normal adults.(ABSTRACT TRUNCATED AT 250 WORDS)

Target polypeptide of a carcinogen is associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes.

Normal rat liver cytosol was found previously to contain a 14,000-dalton polypeptide that is the principal target of the carcinogen N-2-fluorenylacetamide (2-acetyl-aminofluorene) early during hepatocarcinogenesis. By using antiserum that identifies the 14,000-dalton polypeptide in liver cytosol, an immunologically related 17,500-dalton polypeptide was shown to be present in isolated normal liver nuclei, tightly bound to chromatin. We report here that the 14,000-dalton polypeptide is associated with cell multiplication in normal adult hepatocytes. Of 150 hepatocytes in five stages of mitosis, all invariably displayed a greatly increased concentration of the 14,000-dalton polypeptide in cytoplasm, compared to hepatocytes in interphase, by immunostaining with peroxidase-antiperoxidase complex. In contrast, mitosis did not appear to affect the level of the 17,500-dalton polypeptide in nuclei. A small population of high-polyploid hepatocytes with large nuclei had elevated intensities of cytoplasmic immunostain that approached that in mitotic cells. The increased level of discernible 14,000-dalton polypeptide target of the carcinogen in cytoplasm is a marker of the rare mitotic hepatocytes in normal adult rat liver. Ingestion of the liver carcinogen N-2-fluorenylacetamide for 5 wk or an azocarcinogen, 3'-methyl-4-dimethylaminoazobenzene, for 4 wk brought about early foci of hyperplastic hepatocytes in which there was a great overload of detectable 14,000-dalton target polypeptide in their cytoplasm and a near absence of the 17,500-dalton polypeptide in most nuclei. In contrast, the cytoplasm and nuclei in surrounding morphologically nontransformed hepatocytes of livers of the carcinogen-fed rats immunostained to a much smaller degree than in normal adult hepatocytes. Removal of the azo-dye carcinogen for 4 wk resulted in the disappearance of most hyperplastic foci. A few foci did persist and had similar apparent abnormal levels of the two polypeptides. The high concentration of discernible 14,000-dalton target polypeptide in the cytoplasm of hepatocytes in the hyperplastic foci correlates with their known proliferation, whereas the low cytoplasmic level in the surrounding hepatocytes is consistent with the known inhibition of their mitosis by these carcinogens. The collected evidence appears to joint together a chemical carcinogen, a cytoplasmic principal target polypeptide, a chromatin-bound polypeptide, mitosis in normal adult hepatocytes, early and persistent hyperplastic foci caused by carcinogens, and the inhibition of mitosis by carcinogens in surrounding parenchyma in preneoplastic livers.

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.

Persistence of precursor cells of squamous metaplasia in preneoplastic mammary outgrowth lines from mice.

The preneoplastic state is without apparent effect on the induction or prevention of epidermidalization in transplanted mammary outgrowth lines. Development of squamous metaplasia and differentiation (keratinization) were induced in organ cultures of three hyperplastic alveolar and ductular mammary outgrowth lines (D1, MH5, and MH9) that had been extensively passaged in gland-free mammary fat pads of BALB/c virgin mice. The induction was elicited by the mixture of dibutyryl cyclic AMP (0.1 mM), prostaglandins E1, E2, and B1 (each 5 micrograms/ml), and papaverine (1 microM) or by a tenfold higher concentration of dibutyryl cyclic AMP (1 mM) alone for 9 days. The retinoid 2-retinylidene-5,5-dimethyl-1,3-cyclohexanedione at 1 microM and the phorbol ester phorbol 12,13-didecanoate at 10 microM each blocked the induction process. The parameters of the induction and its prevention were analogous in many ways to those previously found with cultures of normal mammary glands of mice and humans, as well as of mouse prostate glands and chick embryo skin. The metaplastic squamous cells that developed in the cultured mammary outgrowths did not proliferate in gland-free mammary fat pads, possibly because the cells were terminally committed or because of insufficient inducers. In contrast, the alveolar and ductular epithelia in the same outgrowths have a transplantable pool of generative cells with the ability to undergo continual proliferation and development. The finding of precursor cells with the potential for epidermoid development and differentiation in the preneoplastic alveolar and ductular outgrowths, despite their extensive serial transplantations, is supportive of the existence of a common or closely associated pool of cells with the ability to develop either into squamous or alveolar mammary epithelium.

Nononcogenic hormone-independent alveoli produced by carcinogens in cultured mouse mammary glands.

Treatment of mouse mammary glands with a high concentration of 7,12-dimethylbenzo(a)anthracene in whole organ culture was reported by Banerjee et al. to transform foci of lobuloalveoli to a hormone-independent state, and to give rise to mammary hyperplastic outgrowths and adenocarcinomas in vivo. In the present study using the identical system, mammary glands of BALB/c mice were exposed to 7,12-dimethylbenzo(a)anthracene or N-2-fluorenylacetamide at low concentrations that bring about maximal incidences of the hormone-independent hyperplastic lobuloalveolar lesions with minimal cytotoxicity. After morphological development of the lobuloalveoli in culture, the glands were enzymatically dissociated into cells and inoculated into gland-free inguinal mammary fat pads of syngeneic mice bearing pituitary gland implants during the initial 8 weeks. After 11 months, fragments of the resultant mammary outgrowths from each mouse were implanted into the gland-free inguinal mammary fat pads of 3 syngeneic mice (not bearing pituitary gland supplements) and were permitted to grow for another 11 months. Mammary outgrowths from the primary and secondary implants were neither neoplastic, anaplastic, nor dysplastic. Also, no hyperplasia in any mammary outgrowth could be attributed to the action of either carcinogen, especially when outgrowths were compared with contralateral outgrowths that arose from the control glands exposed to dimethyl sulfoxide (solvent of the carcinogens) in culture and/or with untreated thoracic mammary glands of the same hosts. One interpretation of these findings is that the hormone-independent, hyperplastic alveolar lesions may not be an appropriate in vitro marker of oncogenic transformation by chemical carcinogens in culture. The great variety of procarcinogens and activated carcinogens that bring about this lesion in vitro and its morphological similarity to presumptive mammary preneoplastic lesions in vivo weigh against this interpretation. A second hypothesis is that high concentrations of procarcinogens, despite their considerable cytotoxicity, complete a multistep process of oncogenic transformation in surviving mammary epithelium, whereas low concentrations optimized to produce the lesions in maximal number do not.

Induction of squamous metaplasia: requirement for cell multiplication, and competition with lobuloalveolar development in cultured mammary glands.

Mouse mammary glands were previously shown to undergo either of two courses of development and differentiation in whole organ culture. The combination of insulin, prolactin, aldosterone, and hydrocortisone induces a structural development of lobuloalveoli, followed by casein production. In the second course, the mixture of dibutyryl cyclic AMP, prostaglandins E1, E2 and B1, and papaverine brings about an extensive squamous metaplasia and excessive keratinization. In the present study, the foci of the metaplastic squamous cells appeared to originate from single or very few cells. A preferential stimulation of squamous cell multiplication was involved in the induction process. Twice the relative number of nuclei incorporated 3H-thymidine in the squamous metaplastic cells than in the surrounding cuboidal epithelium, according to autoradiography. The necessity for cell multiplication was indicated by the reversible and complete inhibitions of both the metaplastic squamous development and 3H-thymidine incorporation by 1 mM hydroxyurea in the culture medium. Simultaneous inductions of both courses of development and differentiation revealed a competitive and reciprocal relationship between the two pathways. The concurrent expressions of both courses were considerably less than those achieved when either pathway was induced alone. Only the combination of the three types of inducers of squamous metaplasia was able to compete effectively with the hormonal induction of lobuloalveolar development and differentiation. The findings suggest that individual metaplastic squamous foci may originate as clones of cells by processes that require cell multiplication, rather than through a direct non- replicative conversion of pre-existent cells of the cuboidal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)