Hamster Kidney Tumor Research Articles

A Novel Therapeutic Regimen to Eradicate Established Solid Tumors with an Effective Induction of Tumor-Specific Immunity

The efficacy of oncolytic viruses depends on multiple actions including direct tumor lysis, modulation of tumor perfusion, and stimulation of tumor-directed immune responses. In this study, we investigated whether a sequential combination of immunologically distinct viruses might enhance antitumor efficacy through the induction of tumor-specific immunity and circumvention or mitigation of antiviral immune responses. The Syrian hamster as an immune-competent model that supports replication of both adenovirus and vaccinia virus was evaluated in vitro and in vivo. The antitumor efficacy of either virus alone or sequential combination of the two viruses was examined in pancreatic and kidney cancer models. The functional mechanism of the regimen developed here was investigated by histopathology, immunohistochemistry staining, CTL assay, and T-cell depletion. The Syrian hamster is a suitable model for assessment of oncolytic adenovirus and vaccinia virus. Three low doses of adenovirus followed by three low doses of vaccinia virus resulted in a superior antitumor efficacy to the reverse combination, or six doses of either virus alone, against pancreatic and kidney tumors in Syrian hamsters. A total of 62.5% of animals bearing either tumor type treated with the sequential combination became tumor-free, accompanied by the induction of effective tumor-specific immunity. This enhanced efficacy was ablated by CD3+ T-cell depletion but was not associated with humoral immunity against the viruses. These findings show that sequential treatment of tumors with oncolytic adenovirus and vaccinia virus is a promising approach for cancer therapy and that T-cell responses play a critical role.

Age, sex and co-exposure to N-ethyl-N-nitrosourea influence mutations in the Alu repeat sequences in diethylstilbestrol-induced kidney tumors in Syrian hamsters.

We report, for the first time, mutations in the Alu repeat regions in the genome of kidney tumors induced by diethylstilbestrol in Syrian hamsters. Among the 66 loci amplified by 11 random primers, 28 loci exhibited insertions, deletions or losses or gains in intensity in the genome of kidney tumor tissues compared with normal kidney tissues from age-matched hamsters. Higher numbers of mutated Alu loci were observed in the tumors of old hamsters compared with young hamsters. In N-ethyl-N-nitrosourea- and diethylstilbestrol-treated hamsters deletion of a 0.59 kb locus amplified with primer OPC03 was observed in most of the female hamsters, but not in male hamsters. An insertion mutation of a 0.498 kb locus amplified with primer OPC03 was observed in 12 of 36 diethylstilbestrol-induced kidney tumors. The cloning and sequencing of the 0.498 kb locus amplified with primer OPC03 revealed that it had significant sequence similarity to the mouse RIKEN cDNA clone. These findings indicate that age, sex and co-exposure to N-ethyl-N-nitrosourea influence mutations in the Alu repeat sequences in the genome of diethylstilbestrol-induced kidney tumors in Syrian hamsters. Structural alterations in Alu repeats in critical target genes may be involved in diethylstilbestrol-induced carcinogenesis.

Somatic mutations in stilbene estrogen-induced Syrian hamster kidney tumors identified by DNA fingerprinting

Kidney tumors from stilbene estrogen (diethylstilbestrol)-treated Syrian hamsters were screened for somatic genetic alterations by Random Amplified Polymorphic DNA-polymerase chain-reaction (RAPD-PCR) fingerprinting. Fingerprints from tumor tissue were generated by single arbitrary primers and compared with fingerprints for normal tissue from the same animal, as well as normal and tumor tissues from different animals. Sixty one of the arbitrary primers amplified 365 loci that contain approximately 476 kbp of the hamster genome. Among these amplified DNA fragments, 44 loci exhibited either qualitative or quantitative differences between the tumor tissues and normal kidney tissues. RAPD-PCR loci showing decreased and increased intensities in tumor tissue DNA relative to control DNA indicate that loci have undergone allelic losses and gains, respectively, in the stilbene estrogen-induced tumor cell genome. The presence or absence of the amplified DNA fragments indicate homozygous insertions or deletions in the kidney tumor DNA compared to the age-matched normal kidney tissue DNA. Seven of 44 mutated loci also were present in the kidney tissues adjacent to tumors (free of macroscopic tumors). The presence of mutated loci in uninvolved (non-tumor) surrounding tissue adjacent to tumors from stilbene estrogen-treated hamsters suggests that these mutations occurred in the early stages of carcinogenesis. The cloning and sequencing of RAPD amplified loci revealed that one mutated locus had significant sequence similarity with the hamster Cyp1A1 gene. The results show the ability of RAPD-PCR to detect and isolate, in a single step, DNA sequences representing genetic alterations in stilbene estrogen-induced cancer cells, including losses of heterozygosity, and homozygous deletion and insertion mutations. RAPD-PCR provides an alternative molecular approach for studying cancer cytogenetics in stilbene estrogen-induced tumors in humans and experimental models. Although the exact functional importance of mutated loci is unknown, this study indicates that these altered loci may participate during tumor progression in the kidney.

Differential oxidant potential of carcinogenic and weakly carcinogenic estrogens: Involvement of metabolic activation and cytochrome P450.

Different estrogens vary in their carcinogenic potential despite having similar hormonal potencies; however, mechanisms of estrogen-induced carcinogenesis remain to be fully elucidated. It has been hypothesized that generation of reactive estrogen-quinones and oxidative stress, both of which result from metabolic activation of estrogens, play an essential role in estrogen-induced carcinogenesis. This hypothesis was tested using the estrogen-receptor (ER)-alpha-positive hamster kidney tumor (H301) and the human breast cancer (MCF-7) cell lines. Estrogens with differing carcinogenic potentials were compared in terms of their capacities to induce 8-iso-prostaglandin F(2alpha) (8- iso-PGF(2alpha)), a marker of oxidative stress. Tumor cells were treated with either 17beta-estradiol (E2), a carcinogenic estrogen or 17-alpha-ethinylestradiol (EE), a weakly-carcinogenic estrogen. Tumor cells were also treated with alpha-naphthoflavone, a cytochrome P450 inhibitor, or a combination of alpha-naphthoflavone and E2 to study the effect of metabolic activation of E2 on E2-induced oxidative stress. H301 cells treated with E2 displayed time- and dose-dependent increases in 8-iso-PGF(2alpha), compared to controls; treatment with 10 nM E2 resulted in a maximal 4-fold induction following 48 h of treatment. In contrast, H301 cells treated with EE did not display an increase in 8-iso-PGF(2alpha) compared with controls. In H301 cells cotreated with alpha-naphthoflavone and E2, alpha-naphthoflavone inhibited the E2-induced increase in 8-iso-PGF(2alpha). These data indicate that a carcinogenic estrogen shows strong oxidant potential, whereas a weakly-carcinogenic estrogen shows poor oxidant potential. Furthermore, inhibiting metabolic activation of a carcinogenic estrogen blocks its oxidant potential. Our data support the hypothesis that metabolic activation and subsequent generation of oxidative stress may play critical roles in estrogen-induced carcinogenesis.

Depletion of mitochondrial DNA and enzyme in estrogen-induced hamster kidney tumors: a rodent model of hormonal carcinogenesis.

Mitochondrial DNA (mtDNA) encodes for 13 polypeptides critical for normal functioning of the electron transport chain and damage to mtDNA has been associated with aging, and implicated in several disease processes. Although damage to mtDNA is being implicated in mutagenesis and carcinogenesis, there are limited studies demonstrating the role and extent of mtDNA damage in human or rodent cancers. Using serial dilution and competitive polymerase chain reaction analysis, we have quantitated the amount of total mtDNA and analyzed the extent of mtDNA damage in estrogen-induced and estrogen-dependent hamster kidney tumors. The hamster kidney tumor model is a useful and widely investigated rodent model of hormonal carcinogenesis, which shares several characteristics with human breast and uterine cancers, and point to a common mechanistic pathway. Our data indicate a significant decrease in the copy number of total mtDNA and the activity of a nuclear-encoded mitochondrial enzyme citrate synthase in hamster kidney tumors compared to age-matched controls. Since there are several hundred mitochondria in a cell and each mitochondrion has multiple copies of mtDNA, a very small percentage of somatic deletion mutation may not be enough to result in a decreased capacity of the mitochondrial genome. However, a significant increase in deletion mutations or a decrease in the mtDNA copy number can result in a decreased oxidative phosphorylation capacity of the mitochondria and decreased energetics, and thus increased susceptibility to the disease process. Therefore, estrogen-induced hamster kidney tumor model can be a useful rodent model of carcinogenesis to understand the role of mtDNA damage in cancer progression and development.

Role of iron in estrogen-induced cancer.

Redox cycling of catecholestrogen metabolites between quinone and catechol forms is a mechanism of generating potentially mutagenic oxygen radicals in estrogen-induced carcinogenesis. Consistent with this concept, multiple forms of oxygen radical-generated DNA damage are induced by estrogen in cell-free systems, in cells in culture and in rodents prone to estrogen-induced cancer. Metal ions, specifically iron, are necessary for the production of hydroxy radicals. Iron has not received much attention in discussions of estrogen-induced carcinogenesis and human hormone-associated cancer, and is the focus of this review. An elevated dietary iron intake enhances the incidence of carcinogen-induced mammary cancer in rats and estrogen-induced kidney tumors in Syrian hamsters. Estrogen administration increases iron accumulation in hamsters and facilitates iron uptake by cells in culture. In humans, elevated body iron storage has been shown to increase the risk of several cancers including breast cancer. A role of iron in hormone-associated cancer in humans offers attractive routes for cancer prevention by regulating metal ion metabolism and interfering with iron accumulation in tissues.

Immunohistochemical analysis of diethylstilbestrol-induced renal tumors in adult male Syrian hamsters: evidence for relationship to peripheral nerve sheath tumors.

Estrogen-induced Syrian hamster kidney tumors (SHKT) are widely used as experimental models for the study of hormonal and renal carcinogenesis. In order to characterize the direction of differentiation of SHKT, kidney sections of diethylstilbestrol (DES)-treated hamsters (1-11 months) were analyzed by immunohistochemistry using a panel of lineage-specific markers. The first tumorous buds found in animals exposed to DES for 4-6 months exhibited prominent S100, Leu-7, and vimentin immunoreactivities. Immunopositivities for neuron-specific enolase, PGP 9.5, desmin, and glial fibrillary acidic protein were mostly detected in medium-sized and large tumors after prolonged exposure to DES (> 6 months). All neoplasms, irrespective of the size and the duration of treatment, appeared negative for cytokeratin, neurofilaments, synaptophysin, and CD99 antibodies. Western blotting confirmed to a large extent the immunohistochemical observations. The systematic analysis of serial kidney sections by confocal microscopy after double immunostaining for S100 and neurofilaments revealed that early neoplastic buds could stem from S100-positive cells associated with nerves bundles. Altogether, these observations suggest that DES-induced SHKT could be related to malignant peripheral nerve sheath tumor and originate from a yet unidentified precursor cell present in the sheath of peripheral nerves.

Demonstration of estrogen receptors and of estrogen responsiveness in the HKT-1097 cell line derived from diethylstilbestrol-induced kidney tumors.

This study was undertaken in order to examine the estrogen sensitivity of HKT-1097, an established cell line recently derived from diethylstilbestrol (DES)-induced kidney tumors in Syrian hamsters. Estrogen receptor (ER) level in HKT-1097, determined by enzyme-linked immunoassay, was 67 fmol/mg protein, i.e., a value approx. 30% lower than that found in Syrian hamster kidney tumors. ER immunostaining in cells fixed with Carnoy's mixture, as well as ER demonstration by Western blotting, suggested DES-induced nuclear translocation or stabilization of the receptor within the nucleus. Kinetic parameters of estrogen binding to ER in HKT-1097 cells were 8.4 x 10(-11) M and 60.8 fmol/mg protein for Kd and Bmax, respectively. The Kd of estrogen binding to ER in HKT-1097 was close to that evaluated for the receptor in breast cancer-derived MCF-7 cell line, whereas the Bmax value was approx. seven times lower in HKT-1097 as compared to MCF-7. In HKT-1097 cells, antiestrogens ICI 182,780 and RU 58,668 induced ER downregulation and competed with estrogen binding to the receptor. As demonstrated by Western blot analysis, DES exposure led to an increased expression of progesterone receptor (PgR) in HKT-1097 cells. Addition of DES to estrogen-free medium produced a stimulation of growth in both HKT-1097 and MCF-7 cells, but the mitogenic effect was less marked for HKT-1097. Despite the fact that ICI 182,780 and RU 58,668 clearly interact with HKT-1097 cell ER, they appeared unable to suppress DES-induced stimulation of growth and increase of PgR expression.

Estrogen mitogenic action. ii. negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications.

In an accompanying report (Moreno-Cuevas, J. E.; Sirbasku, D. A., In Vitro Cell. Dev. Biol.; 2000), we demonstrated 80-fold estrogen mitogenic effects with MTW9/PL2 rat mammary tumor cells in cultures supplemented with charcoal-dextran-treated serum. All sera tested contained an estrogen reversible inhibitor(s). The purpose of this report is to extend those observations to additional sex steroid-responsive human and rodent cell lines. Every line tested showed a biphasic response to hormone-depleted serum. Concentrations of < or = 10% (v/v) promoted substantive growth. At higher concentrations, serum was progressively inhibitory. With estrogen receptor-positive (ER+) human breast cancer cells, rat pituitary tumor cells, and Syrian hamster kidney tumor cells, 50% (v/v) serum caused significant inhibition, which was reversed by very low physiologic concentrations of estrogens. This same pattern was observed with the steroid hormone-responsive LNCaP human prostatic carcinoma cells. Because steroid hormone mitogenic effects are now easily demonstrable using our new methods, the identification of positive results has nullified our original endocrine estromedin hypothesis. We also evaluated autocrine/paracrine growth factor models of estrogen-responsive growth. We asked if insulin-like growth factors I and II, insulin, transforming growth factor alpha, or epidermal growth factor substituted for the positive effects of estrogens. Growth factors did not reverse the serum-caused inhibition. We asked also if transforming growth factor beta (TGFP) substituted for the serum-borne inhibitor. TGFbeta did not substitute. Altogether, our results are most consistent with the concept of a unique serum-borne inhibitor as has been proposed in the estrocolyone model. However, the aspect of the estrocolyone model related to steroid hormone mechanism of action requires more evaluation. The effects of sex steroids at picomolar concentrations may reflect mediation via inhibitor "activated" intracellular signaling pathways.

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5′-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).

Lack of mutations in DNA polymerase beta of estradiol-induced hamster kidney tumors: sequence of hamster DNA polymerase beta cDNA.

We examined the effects of estradiol (E2), the natural estrogenic hormone, on the structure and expression of DNA polymerase beta (DNA pol beta), a DNA repair gene, from E2-induced primary kidney tumors of twelve Syrian hamsters, their metastases, and from kidney tissues surrounding the tumors. We sequenced the coding region of the hamster DNA pol beta and found it to differ from that of the human by 11%. No mutations were detected in the entire coding region including the catalytic domain of the DNA pol beta from E2-induced primary kidney tumors, their metastases, or from kidney tissues surrounding the tumors. The expression of the DNA pol beta mRNA was also not significantly altered in E2-induced kidney tumors or in kidney tissues surrounding the tumors compared to that of control kidney tissues. These results suggest that mutations in the DNA pol beta gene may not be involved in the induction or malignant progression of hamster kidney tumors induced by E2. The nucleotide sequence of the hamster DNA pol beta described here will be useful for the study of the structure and expression of this gene.

Characterization of a cell line established from diethylstilbestrol-induced renal tumors in Syrian hamsters.

This article describes HKT-1097, a new cell line established from renal tumors induced by the protracted administration of diethylstilbestrol (DES) to male Syrian golden hamsters. Cell culture was initiated from tumor samples obtained from two 14-mo.-old animals which had undergone exposure to DES for a period of 11 mo. The HKT-1097 cell line was characterized between Passages 16 and 22 with respect to cell morphology, growth properties, karyology, and the presence of estrogen receptors. Moreover, immunostaining with a panel of antisera was performed to identify the cytological profile of the cell line and establish a parallel with tumor tissue in vivo. HKT-1097 cells are fibroblastoid; their most distinctive feature is that they exhibit strikingly long processes. The HKT-1097 cell line grows as a monolayer with a tendency toward a less stringent density-dependent inhibition of growth. The modal chromosome number is 44, but more than 50% of the cells are aneuploid, suggesting a substantial degree of karyotype instability. HKT-1097 cells express estrogen receptors. They contain immunoreactive vimentin and desmin, but appear negative upon cytokeratin immunostaining. In addition, these cells express glial fibrillary acidic protein and other markers of the neuroectodermal lineage, but lack neurofilament protein. Insofar as the same lineage markers have been demonstrated in DES-induced Syrian hamster kidney tumors (SHKT), we conclude that HKT-1097 cells retain some of the original tumor cell phenotype. The current observations suggest that estrogen-induced SHKT derive from the renal interstitium and point to an involvement of neuroectodermal cells in the development of these neoplasms.

Differential regulation of c-fos expression in estrogen-induced hamster renal tumors compared with kidney not due to creation of an estrogen-response element by point mutation in the gene's flanking sequence

The conversion of a palindromic sequence, GGTCTnnnAGACC, in the 5'-flanking region of the murine c-fos proto-oncogene into a functional estrogen-response element by a single base change into GGTC(A/G)nnnAGACC has previously been postulated [Nawaz et al., 1993] as a possible mechanism of the induction of tumors by estrogens. This attractive hypothesis has been investigated in estradiol-induced Syrian hamster kidneytumors, in H-301 kidney tumor cells (a cell line derived from the Syrian hamster tumor), and in normal kidney tissue. The c-fos gene is differentially regulated by a classical estrogen receptor-mediated process in tumors, whereas in the acutely treated kidney, estradiol induces c-fos expression independent of estrogen-receptor function. In this study, we identified in the 5'-flanking region of the hamster kidney c-fos gene the sequence AGTCCnnnAGACC, which closely resembled but did not appear to function as an estrogen-response element. No mutations were detected in this sequence or in the 5'-flanking region of c-fos genes from three different primary tumors and from H-301 tumor cells. To rule out the possibility of a low copy number of mutant alleles in a tumor sample, polymerase chain reaction-based single-strand conformation polymorphism analysis was performed on 372 base pairs of the 5'-flank of the c-fos gene (-367 to +5 base pairs relative to the transcription start point). Nine different kidney tumor DNA samples and five normal kidney tissue samples (controls) produced an identical pattern of DNA bands, suggesting a lack of natural polymorphisms and mutations in this region of the c-fos gene. Acute treatment of hamsters with 17beta-estradiol for 6 h significantly induced renal c-fos mRNA expression, whereas control levels of c-fos were restored by co-treatment with estradiol and either N-acetyl-L-cysteine or alpha-naphthoflavone. We concluded that the previously observed change in regulatory control of c-fos expression in kidney versus estradiol-induced tumors does not involve the creation of a functional estrogen-response element by single point mutation in the 5'-flanking region of the gene. Additionally, c-fos expression in estradiol-treated hamster kidneys appears to be mediated by free radicals generated by the catechol metabolites of estradiol and not by the activation of any estrogen receptor.

Estrogen, DNA damage and mutations

Estrogen administration to rodents results in various types of DNA damage and ultimately leads to tumors in estrogen-responsive tissues. Yet these hormones have been classified as nonmutagenic, because they did not induce mutations in classical bacterial and mammalian mutation assays. In this review, we have discussed the induction by estrogens of DNA and chromosomal damage and of gene mutations, because the classical assays were designed to uncover mutations only at one specific locus and could not have detected other types of mutations or changes in other genes. Various types of estrogen-induced DNA damage include: (a) direct covalent binding of estrogen quinone metabolites to DNA; (b) enhancement of endogenous DNA adducts by chronic estrogen exposure of rodents; (c) free radical generation by metabolic redox cycling between quinone and hydroquinone forms of estrogens and free radical damage to DNA such as strand breakage, 8-hydroxylation of purine bases of DNA and lipid hydroperoxide-mediated DNA modification. Two different types of chromosomal damage have also been induced by estrogen in vivo and in cells in culture such as numerical chromosomal changes and also structural chromosomal aberrations. Gene mutations have been induced in several cell types in culture either by the parent estrogen or by reactive estrogen quinone metabolites. Furthermore, in estrogen-induced kidney tumors in hamsters, several mutations have been observed in the DNA polymerase β gene mRNA. Estradiol also induces microsatellite instability in these kidney tumors and in premalignant kidney exposed to estradiol. Although this work is still ongoing, it can be concluded that estrogens are complete carcinogens capable of tumor initiation by mutation potentially in critical genes. The hormonal effects of estrogens may complete the development of tumors.

Estrogen-induced microsatellite DNA alterations are associated with Syrian hamster kidney tumorigenesis

Exposure to estrogens is associated with an increase in cancers, including malignancies of the breast and uterus in humans, and of the kidney in hamsters. DNA damage induced by metabolic activation of estrogen has been postulated to result in gene mutations critical for the development of estrogen-induced kidney tumors in hamsters. As part of our examination of the genetic consequences of estrogen-induced DNA damage, we searched for estrogen-induced alterations in microsatellite DNA, a frequent site of mutation in tumors. Genomic DNA isolated from kidney of hamsters treated with estradiol, from estrogen-induced kidney tumors and from untreated age-matched controls, was examined by Southern blot analysis with three multi-locus oligonucleotide probes: (GACA)4, (CAC)6 and (CAG)6. Alterations in DNA fragments containing GACA and CAC tandem repeats were detected in kidney DNA of hamsters treated with hormone for 3 and 4 months, whereas no such effects were seen in control animals. In estrogen-induced tumors, microsatellite alterations were observed in fragments that contain these same two repeat sequences and also CAG repeat sequences. The induction of microsatellite alterations by estradiol in kidney DNA preceding estrogen-induced renal malignancy may play a role in hormone-induced tumorigenesis.

Accumulation of albumin in renal cytosol of hamsters treated with estradiol and in estrogen-induced hamster kidney tumors.

The intracellular accumulation of albumin has been observed in cytosols of benign and malignant human breast tumors and in mammary tumors of rodents induced by carcinogens. Additionally, cellular uptake of albumin has been detected in MCF-7 human breast cancer cells in culture. The clinical relevance of the albumin accumulation in human and rodent mammary tumors is not clear. In this study, we investigated the accumulation of albumin in an estrogen-induced and -dependent hamster kidney tumor model to understand the mechanisms and the role of hormones in this process. Protein accumulation patterns were examined by Western blot analyses in kidney homogenates of hamsters treated with 17beta-estradiol for various lengths of time and in kidney tumors which are induced with 100% incidence by this treatment for at least six months. Such analyses were also carried out in tissues of hamsters treated with the weakly carcinogenic estrogen 17alpha-ethinylestradiol (10% tumor incidence after nine months of treatment). Our data demonstrate the accumulation of albumin in kidney of hamsters treated with 17beta-estradiol but not with 17alpha-ethinylestradiol. Albumin accumulates specifically in the target organ of carcinogenesis, the kidney, however, with no increase in the serum concentrations or in the liver. Tumors do not develop in the livers of hamsters under these conditions of 17beta-estradiol treatment. This accumulation of albumin in hamster kidney may be the result of damage to the glomerulum which may be compromised by estradiol-induced toxicity and therefore unable to filter out excess albumin.

Cytochrome P450 Metabolism of Estradiol in Hamster Liver and Kidney

Estradiol induces kidney tumors in Syrian hamsters. The elevated conversion of estradiol to 4-hydroxylated metabolites in kidney compared to the predominant 2-hydroxylation in liver and other organs, where tumors are not induced by this treatment, has been proposed to be the basis of estrogen-induced carcinogenesis. In this study, we examined the hepatic and renal enzymes catalyzing the formation of catecholestrogens to understand the differences in estrogen metabolism in these organs. In liver, 2-hydroxylation of estradiol is the major metabolic pathway with 4-hydroxylation a minor by-product and with the formation of both catechols responding coordinately to the same inhibitors. Western blot analysis and inhibition studies suggest that the major form catalyzing hepatic estrogen 2-hydroxylation is a member of the CYP3A family, as previously observed with rat liver microsomes, and that 4-hydroxylation is a by-product of this metabolism. In the kidney, 4-hydroxylation of estradiol appears to be catalyzed by more than one enzyme according to the Eadie–Hofstee analysis. Both 2- and 4-hydroxylation in the kidney are affected differentially by inhibitors and are induced by β-napthoflavone. Western blots of renal microsomes reveal that CYP1A2 is induced whereas CYP1A1 is detectable in kidney, but not induced by this treatment. Finally, a part of the 2-hydroxylation and a small part of the 4-hydroxylation by kidney microsomes may be catalyzed by a member of the CYP3A family, since these reactions are partially inhibited by CYP3A inhibitors such as progesterone and other progestins, although renal enzyme levels are much lower than those in the liver as revealed by Western blot. Our data suggest that estrogen 2-hydroxylation in the hamster kidney is catalyzed by members of the CYP1A and CYP3A families, which also contribute to 4-hydroxylation. The majority of 4-hydroxyestradiol formation in the hamster kidney may be catalyzed by a form(s) of the newly discovered CYP1B family that has yet to be characterized.

Immunocytochemical localization of C-myc and C-jun oncoproteins in hamster kidney and estrogen-induced kidney tumors.

The chronic administration of 17beta-estradiol to male Syrian hamsters for 6-7 months induces kidney tumors which express high levels of c-fos, c-myc and c-jun mRNA compared to surrounding tissue or untreated controls. In this study, we have investigated, by immunocytochemical methods, the cellular localization of c-myc and c-jun oncoproteins in estrogen-dependent kidney tumors, in kidney tissue of hamsters treated with 17beta-estradiol for 6 months and in the kidneys of age-matched controls. The c-myc oncoprotein was strongly expressed in tumors, in smooth muscle layers of arteries and in parietal epithelial cells of the glomerulus. In age-matched untreated kidneys, there was little or no staining in the glomerulus, arteries or kidney tubular cells. The c-jun oncoprotein was detected in kidney tumors and in the tubular epithelium of surrounding tissue. The immunoreactivity for c-jun oncoprotein was highest in the tumor, intermediate in estrogen-treated kidney tissue and lowest in kidney tubular cells of controls. It is concluded that the high expression of c-myc in estrogen-induced kidney tumors, in the smooth muscle layer of arteries, and in glomerular parietal epithelial cells in the kidneys of 17beta-estradiol-treated hamsters, but poor expression in control kidneys indicate an involvement of this oncoprotein in the tumorigenic process. In contrast, c-jun is expressed in untreated, in 17beta-estradiol-treated kidneys and in tumors, and may not serve as a prognostic marker in the transformation of these cells to the malignant phenotype.

DIFFERENTIAL REGULATION BY ESTROGEN OF C-FOS IN HAMSTER-KIDNEY AND ESTROGEN-INDUCED KIDNEY TUMOR-CELLS - RECEPTOR MEDIATION VS METABOLIC-ACTIVATION

Chronic administration of 17 beta-estradiol to male Syrian hamsters for at least six months induces estrogen-dependent kidney tumors, which express high levels of c-fos mRNA compared to surrounding renal or control tissues. We have investigated the cellular localization of c-Fos oncoprotein in these tissues and studied the estrogenic regulation of c-fos mRNA in kidney and in H-301 cells, a cell line derived from primary hamster kidney tumors. Immunocytochemical analyses showed that levels of c-Fos protein were high in estrogen-dependent tumors. To study the early events in this model, hamsters were treated with 17 beta-estradiol for only 15 days. At this time, well before the appearance of tumors, c-Fos protein was concentrated in interstitial capillaries, arteries, and podocytes of the glomerulus, but not in renal tubular epithelium, and this pattern was not appreciably changed from controls. To study the regulation of c-fos that led to the altered expression in tumor vs. kidney, total RNA was isolated from kidneys of Syrian hamsters 3-48 h after treatment with single injections of either 17 beta- or 17 alpha-estradiol, 17 alpha-ethinylestradiol, the steroidal antiestrogen ICI 182,780, or 17 beta-estradiol plus ICI 182,780. Similar studies were carried out on H-301 cells grown in D-MEM/F-12 medium and charcoal-stripped serum. The increases in c-fos mRNA levels in H-301 cells but not kidney were elicited by classical estrogen receptor-mediated processes. In H-301 cells, c-fos levels were increased four-fold over controls after 3 h of 17 beta-estradiol treatment and this induction was suppressed by antiestrogen treatment. In hamster kidneys, c-fos levels were increased about two-fold by 17 beta-estradiol, but this induction was not affected by antiestrogen treatment. In H-301 cells but not in hamster kidneys, 17 alpha-ethinylestradiol induced c-fos. 17 alpha-estradiol was more potent than 17 beta-estradiol in the induction of c-fos in hamster kidney but was a poor inducer in H-301 cells. These studies are consistent with regulation of c-fos expression in H-301 tumor cells by an estrogen receptor-mediated mechanism. But in hamster kidney, c-fos expression does not appear to be regulated by receptor-mediated pathways. We propose that it may be induced by estrogen metabolites.

DIFFERENTIAL EXPRESSION OF NEU MESSENGER-RNA AND PROTEIN IN HAMSTER-KIDNEY AND ESTROGEN-INDUCED KIDNEY TUMORS

An inverse relationship between the expression of the neu oncogene and estrogen receptors has been observed in breast cancer patients. In this study, we examined neu expression in the estrogen-induced and -dependent hamster kidney tumors, in kidney and in controls to evaluate the usefulness of this animal model far studying the regulation of genes important in hormonal cancer. The expression of neu mRNA was analyzed by Northern analysis and Neu oncoprotein localization by immunocytochemistry. The Neu oncoprotein was detected in several segments of proximal and distal kidney tubules, the loops of Henle and the parietal epithelial cells of Bowman's capsule but not in the tumor. neu mRNA was expressed in renal tissue after estrogen treatment and in untreated controls, but not in kidney tumors. The absence of Neu oncoprotein and mRNA from those cell types that have previously been shown to overexpress estrogen receptors in response to estrogen, suggests that the estrogen receptor and neu genes are interdependent in this tumor system, which may thus be a useful animal model for studying the regulation of neu by estrogens.