NFkappaB prevents apoptosis and liver dysfunction during liver regeneration.

Although NFkappaB binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NFkappaB during liver regeneration by delivering to the liver a superrepressor of NFkappaB activity using an adenoviral vector expressing a mutated form of IkappaBalpha. This adenovirus (Ad5IkappaB) was almost exclusively expressed in the liver and inhibited NFkappaB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5IkappaB, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IkappaB but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5IkappaB but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFkappaB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.

Phenotypic modulation during tumorigenesis by clones of transformed rat liver epithelial cells.

From nine clonal subpopulations (strains) of chemically transformed cultured rat hepatic epithelial cells which were tumorigenic when implanted into 1-day-old isogeneic rats, a cell line was reestablished from each tumor and the cellular properties of the tumor-derived cell lines were compared to those of the corresponding progenitor cells that were implanted to produce the tumors. In seven of eight instances, the cellular DNA content of the tumor-derived cells was virtually identical to the DNA content of the respective progenitor cells, but in one case the tumor cells had twice as much DNA as did their progenitor cells. During the development of tumors in vivo, other cellular phenotypic properties often underwent considerable, but variable changes. These changes included the activity of gamma-glutamyl transpeptidase, the growth properties on plastic surfaces, and the expression of LDH isozymes. Although there was a relative enhancement in the ability of most of the tumor-derived cells to proliferate or to form colonies in calcium-poor medium, several tumor-derived cell lines had very low colony-forming efficiencies in media containing either normal or low levels of calcium. The most consistent association between phenotypes expressed in vitro and tumorigenicity was the ability of cells to form colonies in soft agar; all tumor-derived lines expressed this phenotype, and with some of them this phenotype was acquired only during the process of tumor formation in vivo. These results demonstrate that further phenotypic and genotypic alterations may occur in vivo during tumor formation by chemically transformed cultured cells following their implantation into isogeneic animals; and some of the alterations that occur in vivo may be necessary for the complete expression of tumorigenicity. Although anchorage-independent growth capacity cannot be used to predict the tumorigenicity of clones of rat liver epithelial cells chemically transformed in vitro, this growth property appears to be invariably induced prior to or during the formation of tumors in vivo by these cells.

Transforming growth factors produced by normal and neoplastically transformed rat liver epithelial cells in culture.

The secretion of transforming growth factors (TGFs) alpha and beta by normal, chemically transformed, and malignant rat liver epithelial cell lines was investigated. The WB-F344 normal cultured rat liver epithelial cell line does not secrete an epidermal growth factor-like (putatively TGF-alpha) activity, but several clonal cell strains derived from WB-F344 cells which had been treated with N-methyl-N'-nitro-N-nitrosoguanidine, especially those that expressed high levels of gamma-glutamyl transpeptidase, secreted TGF-alpha-like activity into their conditioned media. Cell lines obtained from tumors which were produced by these cell strains varied in their abilities to secrete TGF-alpha, even though they all expressed high levels of gamma-glutamyl transpeptidase activity. When two of the non-TGF-alpha-secreting tumor cell lines were transplanted into isogeneic rats, the tumors that formed contained high levels of TGF-alpha-like activity. Although epidermal growth-factor (hence, TGF-alpha also) inhibited the proliferation of several of these tumor cell lines in monolayer cultures, this growth factor often paradoxically stimulated the anchorage-independent growth of the same cell lines. In contrast to TGF-alpha-like activity, all cell lines/strains released TGF-beta activity into their conditioned media. However, while both normal or chemically transformed cell strains typically produced the inactive form of TGF-beta, the tumor cell lines tended to produce activated TGF-beta de novo. Anchorage-independent growth of cell lines that produced active TGF-beta was either stimulated, inhibited, or unaffected by TGF-beta. Cell lines that were inhibited by TGF-beta concurrently produced TGF-alpha which was usually able to overcome the negative "autocrine" effect of TGF-beta. We conclude that both TGF-alpha and TGF-beta, singly or in combination, are variously involved in the growth of transformed rat liver epithelial cells. TGF-alpha has a predominantly positive autocrine action on the growth of rat liver epithelial tumor cell lines. The "paracrine" effect of TGF-beta may be at least as important as its autocrine effect in the growth of these transformed epithelial cell lines.

Clonal analysis of the expression of multiple transformation phenotypes and tumorigenicity by morphologically transformed 10T1/2 cells.

Seventy-five clonal populations of morphologically transformed 10T1/2 cells were established from independent Type II and Type III foci that were of spontaneous origin or were induced by the carcinogenic agents N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene diol epoxide-I, and 3-methylcholanthrene. Clonal populations were characterized for expression of selected transformation phenotypes, including growth to elevated saturation density before cessation of proliferation, anchorage independence, ability to reconstruct foci when plated in the presence of wild-type 10T1/2 cells, and tumorigenicity. Forty-one % of the clonal populations expressed only the phenotype of morphological transformation, while 20% expressed all of the transformation phenotypes, including tumorigenicity, in addition to morphological transformation. The remaining clonal populations expressed varying combinations of one or more of the four transformation phenotypes. Clonal populations expressing almost all of the 16 possible combinations of the transformation phenotypes were observed, suggesting that the individual phenotypes segregated independently. Morphological transformation alone was a poor indicator of tumorigenicity, correctly predicting the tumorigenic potential of only 37% of the clonal populations. Among morphologically transformed clonal populations, coexpression of anchorage independence correctly predicted the tumorigenicity of 81% and coexpression of reconstruction of foci on a confluent lawn of wild-type cells correctly predicted the tumorigenicity of 91%. The probability that a morphologically transformed clonal population was tumorigenic correlated with the total number of transformation phenotypes expressed. Expression of the transformation phenotypes differed between tumorigenic and nontumorigenic clonal populations but not between clonal populations established from Type II and Type III foci. Tumorigenicity varied among transformed clonal populations that were induced by the different carcinogenic agents or were of spontaneous origin but did not differ between clonal populations established from Type II and Type III foci.

Colony-forming ability in calcium-poor medium in vitro and tumorigenicity in vivo not coupled in clones of transformed rat hepatic epithelial cells.

The ability of eukaryotic cells in culture to proliferate in calcium-poor medium has been found to characterize populations of transformed cells, but the relationship between this phenotypic property and tumorigenicity at the cellular level is unclear. Thus, we have isolated 14 clonal subpopulations, based on their ability to colonize in calcium-poor medium, from a parental tumorigenic rat hepatic epithelial cell line which was transformed by multiple exposures to N-methyl-N'-nitro-N-nitrosoguanidine. These clonal subpopulations of cells were tested for their ability to grow in soft agar, to express gamma-glutamyl transpeptidase activity, and to form tumors upon back-transplantation into isogeneic newborn rats. The results indicated that clonal subpopulations of cells selected by their ability to grow in calcium-poor medium were phenotypically heterogeneous for gamma-glutamyl transpeptidase activity and anchorage-independent growth, and, more importantly, they were not more tumorigenic than the phenotypically heterogeneous parental cell line. This observation suggests that the capability of cultured hepatic epithelial cells to grow in calcium-poor medium is not tightly coupled to the tumorigenic phenotype.

Clonal analysis of tumorigenicity and paratumorigenic phenotypes in rat liver epithelial cells chemically transformed in vitro.

Clonal subpopulations of a chemically induced tumorigenic rat liver epithelial cell line were analyzed for their cellular, biochemical, and in vitro growth properties and their tumorigenicity after injection into day-old newborn isogeneic rats. The phenotypic properties studied included DNA content; growth rate in culture; activities of gamma-glutamyl transpeptidase, NADH diaphorase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase; ability to grow in calcium-poor medium; and ability to form colonies in soft agar. The results show that none of these phenotypes cosegregates with tumorigenicity and therefore is not reliable as a "marker" phenotype for neoplastic transformation in cultured rat liver epithelial cells. The poor correlations, either qualitatively or quantitatively, between paratumorigenic phenotypes and tumorigenicity suggest that neoplastic transformation in these cells involves a specific transforming gene locus or loci and that in vitro paratumorigenic phenotypes are merely epiphenomena of neoplastic transformation and progression. This study further reveals that the efficiency of the tumorigenicity assay of cultured rat liver epithelial cells in isogeneic newborn rats can be considerably improved by incubating the cells in medium containing only trace amounts of serum prior to transplantation into the host animals.

Cycle-dependent removal of certain methylated bases from DNA of 10T1/2 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The loss of N-methyl-N-nitro-N-nitrosoguanidine (MNNG)-induced adducts from DNA was quantitated during the G1 and S phases of synchronously proliferating mouse 10T1/2 cells exposed to MNNG (2 microgram/ml) when they were in either confluence-induced arrest of proliferation or at the G2-S border. When treated at either time, N-7-methylguanine and O6-methylguanine were not excised from the template DNA during the subsequent S phase. However, both lesions were efficiently removed during the G1 phase immediately following exposure to MNNG, as well as during the second G2 phase after an S phase during which no loss occurrred. In contrast, N-3-methyladenine was lost rapidly during both the G1 and S phases following MNNG treatment. N-7 Methylguanine and O6-methylguanine were removed from logarithmically growing cell populations more slowly than from cells passing synchronously through the G1 phase. However, when the observed rate of loss in logarithmic cultures was corrected for the fraction of the logarithmic population located in the S phase, the rates of loss of the two methylated bases were the same as those observed in the G1 phase in synchronous cultures.

Gradation of carcinogen-induced capacity for anchorage-independent growth in cultured rat liver epithelial cells.

The effect of epidermal growth factor (EGF) on the capacity for anchorage-independent growth of chemically treated rat hepatic epithelial cells has been investigated. We have performed the studies using 16 clonally derived cell strains which represented single cell-derived subpopulations of a heterogeneous rat hepatic epithelial cell line that had been tumorigenically transformed by 11 repeated treatments with N-methyl-N'-nitro-N-nitrosoguanidine. The results can be summarized as follows. (a) Secondary clonal subpopulations isolated from the colonies formed by these strains in soft agar subsequently and invariably acquired markedly enhanced colony-forming efficiencies as compared to their parental strains. (b) EGF could enhance or induce colony-forming ability in soft agar in all of these cell strains. (c) The magnitudes of enhancement of the colony-forming efficiencies by EGF in soft agar could not be correlated with the absolute EGF-binding capacity of these cell strains. (d) The enhancement or induction of the colony-forming ability by EGF was either reversible or irreversible, partially correlating with the expression of gamma-glutamyl transpeptidase activity by the strains. These findings indicate that the cellular capacity of liver epithelial cells to grow anchorage independently in soft agar medium can be graded according to the pattern of response of EGF induction of colony-forming ability. These grades may reflect the level of neoplastic transformation of these cells. Moreover during the multistep transformation of rat hepatic epithelial cells by a chemical carcinogen, EGF can be used to reveal the presence of altered cells which have acquired partial capacity for the anchor-age-independent growth property. This property may constitute an additional identifiable early step of the neoplastic transformation of these cells.

Clonal isolation of populations of gamma-glutamyl transpeptidase-positive and -negative cells from rat liver epithelial cells chemically transformed in vitro.

In a population of cultured rat liver epithelial cells transformed by 11 brief treatments with N-methyl-N'-nitro-N-nitrosoguanidine, 9% of the cells stained intensely for gamma-glutamyl transpeptidase (GGT). We have isolated from this phenotypically heterogeneous tumorigenic cell population 11 GGT-positive and 7 GGT-negative clonal subpopulations (from single cells) and have analyzed the ploidy and selected biochemical, histochemical, and growth properties of the cells in these clonal sublines. As compared to the GGT-negative strains and normal diploid rat liver epithelial cells, cells of the GGT-positive strains are larger in size, have greater DNA content, proliferate more slowly in culture, and have higher specific activities of NADH diaphorase, glucose-6-phosphate dehydrogenase, pyruvate kinase, and lactate dehydrogenase. The GGT-positive strains also show greater alteration and heterogeneity than do the GGT-negative strains in their ability to store glycogen and in their expression of lactate dehydrogenase isozymes. The results indicate that enzymatic changes commonly observed in "altered" hepatocytes in rat livers exposed to chemical carcinogens in vivo can also be produced in vitro in cultured hepatic epithelial cells by treatment with carcinogens. Moreover, treatment of a cell line with a chemical carcinogen generates a population of cells vastly heterogeneous in both their phenotypes and genotypes. Isolation of clonal subpopulations from the resulting cell line allows critical examination of the linkage and mechanistic relationship between tumorigenicity and many paratumorigenic phenotypes.

Regulation of transforming growth factor alpha messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol ester and hormones.

Transforming growth factor alpha (TGF-alpha) is produced by many transformed cells, but little is known about the regulation of its expression. We examined TGF-alpha mRNA levels in a set of cloned neoplastic cell lines derived by chemical transformation of a normal rat liver epithelial cell. The untransformed parental cell line, WB-344, did not express a detectable level of TGF-alpha mRNA, whereas GP6ac, a transformed line capable of autonomous growth in soft agar, expressed TGF-alpha. When GP6ac cells were treated with agents thought to regulate protein kinase C activity, e.g., the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF-alpha mRNA levels increased by 8- to 11-fold. The induction of TGF-alpha mRNA was detectable at 2 h, was maximal at 8-12 h, and declined by 24 h. Angiotensin, bradykinin, epinephrine, and epidermal growth factor also increased TGF-alpha mRNA by 2- to 5-fold. In contrast, parental WB cells neither expressed TGF-alpha mRNA, nor responded to TPA. TPA also increased epidermal growth factor receptor mRNA in GP6ac cells but the effect was less prolonged; maximal levels were seen at 4 h after TPA exposure and returned to control levels by 12 h. TPA increased TGF-alpha mRNA in GP6ac cells, in part, by increasing transcription of the TGF-alpha gene as measured by run-on transcription rates in isolated nuclei. In addition, the induction of TGF-alpha by TPA was blocked by concurrent incubation with agents that inhibit protein synthesis. However, if TPA was present for at least 2 h, subsequent addition of cycloheximide enhanced the effect of TPA. This indicates that the induction of TGF-alpha in GP6ac cells is comprised of at least two phases demarcated by the requirement for protein synthesis. The time course of induction and the sensitivity to inhibition of protein synthesis distinguish the effect of TPA on TGF-alpha mRNA from that of other genes regulated by TPA, e.g., c-myc and c-fos. These data also suggest that chemical transformation of rat liver epithelial cells leads to expression of TGF-alpha mRNA, and that once expressed, TGF-alpha mRNA can be modulated in a protein kinase C-dependent manner.

Age-dependent induction of hepatic tumor regression by the tissue microenvironment after transplantation of neoplastically transformed rat liver epithelial cells into the liver.

Age is the biggest risk factor associated with the development of cancer. Whereas the incidence of neoplastic disease increases dramatically in aging humans and experimental animals, the effects of aging on tumorigenesis are poorly understood. Using a rodent model, we have previously shown that the microenvironment of the hepatic parenchyma regulates hepatic tumor formation from transplanted neoplastic cells in an age-dependent manner. In the current study, we have investigated the mechanistic basis for the age-dependent suppression of tumor formation by transplanted BAG2-GN6TF rat liver epithelial tumor cells. Examination of liver tissue at 7 and 14 days after transplantation of liver tumor cells revealed the presence of injection-site tumors in both young and old animals. With time, these tumors spontaneously regressed from young adult livers, leaving no tumor remnant and without evidence of injury to the parenchyma. In contrast, tumors detected in old animals at early time points after transplantation persisted for the remainder of the life of the host. Reduced cell proliferation and increased apoptotic cell death were detected in hepatic tumors in young rats relative to hepatic tumors in old rats. These observations suggest that the regression of hepatic tumors from young rats was the direct result of an increased ratio of cell death to cell birth, whereas the persistence and expansion of hepatic tumors in old rats was related to increased cell proliferation relative to cell death. Because young adult rats developed persistent (nonregressing) tumors after transplantation of BAG2-GN6TF cells to extrahepatic sites, the consistent regression of BAG2-GN6TF tumors from livers of young rats seemed to be largely a result of interactions between tumor cells and factors specific to the liver microenvironment. These data indicate that the hepatic microenvironment of young rats can negatively regulate the growth of transformed liver epithelial cells, but with increasing age, the ability of the hepatic microenvironment to suppress the growth of neoplastic tissue deteriorates. Age-associated alterations in tissue microenvironments may thus permit the development of tumors late in life.

Induction of foci of morphologically transformed cells in synchronized populations of 10T1/2 cells by N-methyl-N'-nitro-N-nitrosoguanidine and the effect of spontaneous transformation on calculated transformation frequency.

Exposure of synchronized C3H10T1/2 (clone 8) cell populations of various sizes to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at a concentration of 2 micrograms/ml for 30 min at 24 h after release from confluence-induced arrest of proliferation produced neoplastic transformation (formation of foci of morphologically altered cells) by a random but episodic process in a small fraction of the cells at risk soon after treatment. The fraction of dishes that contained type II or type III foci increased as the number of cells at risk increased. In contrast, the development of spontaneous foci is a stochastic process that depends on the number of new cells that form during population growth and is independent of the number of cells that are plated (J. W. Grisham et al., Cancer Res., 48: 5969-5976,1988). When there were small numbers of cells at risk, spontaneous formation of foci was a source of considerable error in evaluating MNNG-induced transformation frequency. In surviving cell populations of less than 1000-3000 cells/100-mm dish, the frequency of induction of foci by MNNG could not be distinguished statistically from the frequency with which foci were expected to form spontaneously. When the fraction of MNNG-treated dishes that contained foci was adjusted for the fraction of pooled control dishes that contained foci, the number of foci induced by a uniform dose of MNNG was found to vary with the number of surviving cells. However, the MNNG-induced transformation frequencies calculated by the Poisson method were independent of the size of the population of cells at risk, provided the population of cells at risk was of sufficient size to allow spontaneous and induced transformation to be distinguished statistically. The results of this study show that the frequency of MNNG-induced transformation can be quantitated in cultures of 10T1/2 cells that contain varying but sufficient numbers of cells at risk when spontaneous transformation is considered. Furthermore, these observations suggest that MNNG-induced transformation of 10T1/2 cells occurs with the frequency and characteristics of a mutation-like change involving a single gene.

Spontaneous formation of foci of morphologically transformed cells in populations of C3H 10T1/2 (clone 8) cells.

Spontaneous formation of morphologically altered foci of types II and III (neoplastic transformation) was examined in populations of C3H 10T1/2 (10T1/2) cells. Initial surviving cell densities ranged from 3 to 3 x 10(5) cells/100-mm dish and the final cell density was approximately 2 x 10(6) cells/dish, yielding widely differing numbers of population doublings but similar numbers of cell births from the time of cell plating to the attainment of confluence. Spontaneous formation of foci was independent of the initial surviving cell densities (and, therefore, of the number of population doublings) but was related to the number of cell divisions (cell births) between the time the cell population was plated and when suppression of proliferation of wild-type cells occurred in confluent cultures. In 418 pooled asynchronously proliferating cultures in 100-mm dishes the 95% confidence limits for the fraction of dishes containing foci was 0.041-0.089 for type II foci and 0.008-0.036 for type III foci; for cell populations in 2041 pooled cultures in 100-mm dishes, the proliferation of which was synchronized by release from confluence-induced arrest of proliferation, the 95% confidence limits for the fraction of dishes containing foci were 0.150-0.166 for type II foci and 0.017-0.032 for type III foci. Using the Poisson method, the 95% confidence limits for rates of spontaneous transformation in asynchronously proliferating populations of 10T1/2 cells were 1.4-3.2 x 10(-8)/cell/division for type II foci and 0.28 to 1.3 x 10(-8)/cell/division for type III foci; in populations in which proliferation was initially synchronized by release from confluence-induced arrest, spontaneous transformation rates were 5.6-6.3 x 10(-8)/cell/division for type II foci and 0.59-1.1 x 10(-8)/cell/division for type III foci. Spontaneous transformation occurred in populations of wild-type 10T1/2 cells at the rates and with the characteristics expected of the mutation of a single gene locus.

Clonal cosegregation of tumorigenicity with overexpression of c-myc and transforming growth factor alpha genes in chemically transformed rat liver epithelial cells.

Tumorigenicity was correlated with levels of expression of the genes for transforming growth factor alpha (TGF-alpha), epidermal growth factor receptor, c-myc, c-H-ras, and c-K-ras in a series of 16 clonally derived transformed liver epithelial cell lines. The clonal lines, which varied in tumorigenicity from 0 to 97%, were established from a phenotypically heterogeneous population produced by repeated exposure of diploid WB-F344 (WB) cells to N-methyl-N'-nitro-N-nitrosoguanidine. Segregation of gene expression with tumorigenicity among clonal lines was determined by correlating rank orders of gene expression by clones relative to expression by wild-type WB cells. Only the expression of the c-myc gene correlated with tumorigenicity among all transformed clones. TGF-alpha gene expression was not correlated with tumorigenicity among all clones, but it was highly correlated with tumorigenicity among clones that expressed the c-myc gene above the median level for all clones (greater than 5-fold the level of expression by WB cells). Even high levels of expression of the TGF-alpha gene (up to 60-fold the level of expression by WB cells) were not correlated with tumorigenicity among the clones expressing the c-myc gene at levels less than 5-fold the level of expression by WB cells. Clones which simultaneously overexpressed both c-myc and TGF-alpha genes at levels above the median levels for all clones were significantly more tumorigenic than were clones which expressed either or both genes at lower than median levels. These results suggest that overexpressed c-myc and TGF-alpha genes cooperate in their association with tumorigenicity. Most of the highly tumorigenic clones that overexpressed c-myc and TGF-alpha also overexpressed the c-H-ras and/or the c-K-ras genes; clones that overexpressed neither of the c-ras genes nor the genes for c-myc and TGF-alpha were not very tumorigenic, while clones that expressed one or both c-ras genes (but not both c-myc and TGF-alpha) were variably tumorigenic over an intermediate range.

In vitro assay of cytotoxicity with cultured liver: accomplishments and possibilities.

Tissue cultures offer potential advantages for assaying the toxicity of chemicals and for evaluating tissue susceptibility to toxic agents. Several properties of cultured cells hinder the immediate, widespread use of tissue cultures to assay toxicity routinely. These points are illustrated by briefly reviewing attempts to utilize different types of hepatic cultures to evaluate the actions of carcinogenic chemicals in vitro. Hepatocytes in vivo apparently can metabolize all known procarcinogenic chemicals, but the process of tissue isolation and the environmental conditions in vitro may modify drastically the responses of hepatocytes and other cultured hepatic cells to toxic chemicals. Before cell cultures can be used routinely as the basis of screening systems to detect chemical toxins, specificity and sensitivity of response to chemicals representing all chemical classes must be validated by laboratory studies.

Induction of rat WT1 gene expression correlates with human chromosome 11p11.2-p12-mediated suppression of tumorigenicity in rat liver epithelial tumor cell lines.

We have previously identified and mapped a locus within human chromosome 11p11.2-p12 that suppresses the tumorigenic potential of some rat liver tumor cell lines. In the present study, possible molecular mechanisms of human 11p11.2-p12-mediated liver tumor suppression were investigated by examining gene expression patterns in suppressed and non-suppressed microcell hybrid (MCH) cell lines. The parental rat liver tumor cell lines (GN6TF and GP7TB) express moderate levels of p53 mRNA and protein, overexpress mRNAs for c-H-ras, c-myc, and TGFá, and do not express detectable levels of WT1 mRNA or protein. Suppression of tumorigenicity by human chromosome 11p11.2-p12 was not accompanied by significant alterations in the levels of expression of p53, c-myc, or TGFá. Expression of c-H-ras was decreased significantly in both suppressed and non-suppressed MCH cell lines, suggesting that down-regulation of c-H-ras is not directly responsible for tumor suppression. In contrast, the level of expression of WT1 correlated precisely with tumor suppression in this model system. All suppressed MCH cell lines expressed WT1 mRNA and protein at levels comparable to that of untransformed rat liver epithelial cells (WB-F344), whereas only trace WT1 mRNA and protein were detected in a non-suppressed MCH cell line. PCR analysis demonstrated that two suppressed MCH cell lines do not carry the human WT1 gene, indicating that WT1 expression in these lines originates from the rat locus. Furthermore, RT-PCR analysis showed that each of the four known splice variants of the WT1 mRNA are expressed in these suppressed MCH cell lines, recapitulating the expression pattern observed in the untransformed rat liver epithelial cells. Re-expression of tumorigenicity by suppressed MCH cell lines was accompanied by the coordinate loss of human chromosome 11p11.2-p12 and of WT1 gene expression, suggesting that one or more human 11p11.2-p12 genes are required for sustained expression of WT1 in these cell lines. Together, these results suggest that the molecular mechanism governing human chromosome 11p11.2-p12-mediated liver tumor suppression may involve induction of rat WT1 gene expression under the direct or indirect transcriptional regulation of a genetic locus (or loci) on human 11p11.2-p12.

Cell types in long-term propagable cultures of rat liver.

Under the conditions of this experiment, propagable hepatic epithelial cells in long-term culture were derived from a small, nonparenchymal precursor cell present in the liver in vivo. Progeny of the nonparenchymal precursor cell weakly expressed some hepatocyte-like properties in culture. Hepatocytes were not clonogenic under the conditions of this experiment. Propagable hepatic epithelial cells in long-term culture probably originate from hepatic stem cells in vivo. Terminal biliary ductular cells are facultative stem cells for hepatocytes.

Mutagenic potency at the Na+/K+ ATPase locus correlates with cycle-dependent killing of 10T1/2 cells.

Perturbation of DNA replication by chemical-DNA adducts produced by exposure to mutagenic/carcinogenic chemicals results in mutagenic or cytotoxic damage in the DNA. Demonstration of a correlation between cell cycle dependency of cytotoxicity and point mutation at the Na+/K+ ATPase gene could suggest that the two consequences of chemical exposure are caused by the same damage in the template DNA and that both are mediated through DNA replication-associated mechanisms. N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N'-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, and benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide demonstrated cell cycle-related patterns of cytotoxicity in 10T1/2 cells, with maximal cell killing produced by exposure in early S phase, and were highly efficient mutagens of the Na+/K+ ATPase gene relative to their cytotoxic potential. In contrast, methyl methanesulfonate and N-acetoxy-N-2-fluorenylacetamide were maximally cytotoxic in cell populations exposed in early G1 phase and were weak mutagens of the Na+/K+ ATPase gene at comparable levels of cytotoxicity. These data suggest that mutagenic/carcinogenic chemicals that are effective at producing mutations by misreplication kill cells by a related mechanism that may be associated with the perturbation of DNA replication.

Cytotoxicity of monofunctional alkylating agents. Methyl methanesulfonate and methyl-N'-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN1 and SN2 mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O6 position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect is potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS, MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMs kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.

Effects of 4-nitroquinoline-1-oxide on population growth, cell-cycle compartmentalization and viability in human lymphoblastoid cells.

The mechanism by which the carcinogen 4-nitroquinoline-1-oxide (4-NQO) kills mammalian cells is unclear; however, damage to DNA is presumed to be involved. We examined the relationship between the kinetics of cell death and alteration of cell-cycle compartmentalization after exposure of T5-1 human lymphoblastoid cells to 4-NQO (50 to 500 ng/ml) to establish whether cytotoxicity was related to the perturbation of DNA replication. Dose-dependent reductions in cell proliferation and cell viability were present from 1 day after treatment. Maximal reductions in viability were observed 2 days after exposure. Concentrations of 4-NQO of up to 175 ng/ml did not affect cell-cycle compartmentalization, but 250 ng 4-NQO/ml caused a transient accumulation of cells in S phase after 1 day. Only after 500 ng 4-NQO/ml was a marked and prolonged S-phase block observed from 1 day onwards. The lack of a strong correlation between S-phase block and cell death after exposure to 4-NQO suggests that responses to DNA damage in addition to perturbation of DNA replication entrain 4-NQO-induced cell lethality.