Transforming growth factor-beta1 is a new form of tumor suppressor with true haploid insufficiency.

Components of the transforming growth factor-beta (TGF-beta) signal pathway function as classic tumor suppressors, but the role of the TGF-betas themselves is less clear. Here we show that mice heterozygous for deletion of the TGF-beta1 gene express only 10-30% of wild-type TGF-beta1 protein levels. Although grossly normal, these mice have a subtly altered proliferative phenotype, with increased cell turnover in the liver and lung. Treatment of these mice with chemical carcinogens resulted in enhanced tumorigenesis when compared with wild-type littermates. However, tumors in the heterozygous mice did not lose the remaining wild-type TGF-beta1 allele, indicating that the TGF-beta1 ligand is a new form of tumor suppressor that shows true haploid insufficiency in its ability to protect against tumorigenesis.

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Fever and survival.

The significance of fever in response to a bacterial infection has been investigated using the lizard Dipsosaurus dorsalis as an animal model. These lizards develop a fever of about 2 degrees C after injection with the bacterium Aeromonas hydrophila. To determine whether this elevation in body temperature increases the resistance of the host to this infection, as measured by survival, lizards were infected with the live bacteria and placed in a neutral (38 degrees C), low (34 degrees or 36 degrees C), or high (40 degrees or 42 degrees C) ambient temperature. An elevation in temperature following experimental bacterial infection results in a significant increase in host survival.

Immunologic and hematopoietic effects of CD40 stimulation after syngeneic bone marrow transplantation in mice.

CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.

The p53 response to DNA damage in vivo is independent of DNA-dependent protein kinase.

Ionizing radiation (IR) exposure causes mammalian cells to undergo p53-dependent cell cycle arrest and/or apoptosis. The in vivo role of DNA-dependent protein kinase (DNA-PK) in the transduction of the DNA damage signal to p53 remains unresolved. To determine the relationship between DNA-PK and p53, we studied the cell cycle and apoptotic responses to IR in mice deficient in DNA-PK. Using the slip mouse, which harbors an inactivating mutation of the DNA-PK catalytic subunit (DNA-PKcs), we demonstrated not only that these DNA-PKcs null mutants were highly radiosensitive but also that upon IR treatment, p53 accumulated in their cultured cells and tissue. Induced p53 was transcriptionally active and mediated the induction of p21 and Bax in slip cells. Examination of the thymic cell cycle response to IR treatment indicated that the slip G(1)/S-phase cell cycle checkpoint function was intact. We further show that slip mice exhibited a higher level of spontaneous thymic apoptosis as well as a more robust apoptotic response to IR than wild-type mice. Together, these data demonstrate that the p53-mediated response to DNA damage is intact in cells devoid of DNA-PK activity and suggest that other kinases, such as the product of the gene (ATM) mutated in ataxia telangiectasia, are better candidates for regulating IR-induced phosphorylation and accumulation of p53.

Gadd45a acts as a modifier locus for lymphoblastic lymphoma.

GADD:45a-/- and p53-/- mice and cells derived from them share similar phenotypes, most notably genomic instability. However, p53-/- mice rapidly develop a variety of neoplasms, while Gadd45a-/- mice do not. The two proteins are involved in a regulatory feedback loop, whereby each can increase the expression or activity of the other, suggesting that common phenotypes might result from similar molecular mechanisms. Mice lacking both genes were generated to address this issue. Gadd45a-/-p53-/- mice developed tumors with a latency similar to that of tumor-prone p53-/- mice. However, while p53-/- mice developed a variety of tumor types, nearly all Gadd45a-/-p53-/- mice developed lymphoblastic lymphoma (LBL), often accompanied by mediastinal masses as is common in human patients with this tumor type. Deletion of Gadd45a in leukemia/lymphoma-prone AKR mice decreased the latency for LBL. These results indicate that Gadd45a may act as modifier locus for T-cell LBL, whereby deletion of Gadd45a enhances development of this tumor type in susceptible mice. Gadd45a is localized to 1p31.1, and 1p abnormalities have been described in T-cell lymphomas. Related human tumor samples did not show Gadd45a deletion or mutation, although changes in expression could not be ruled out.

Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species.

BACKGROUND: In the autumn of 1992, a novel form of chronic, active hepatitis of unknown etiology was discovered in mice at the National Cancer Institute-Frederick Cancer Research and Development Center (NCI-FCRDC), Frederick, Md. A high incidence of hepatocellular tumors occurred in affected animals. The disease entity was originally identified in A/JCr mice that were untreated controls in a long-term toxicologic study. PURPOSE: Our original purpose was to determine the origin and etiology of the chronic hepatitis and to quantify its association with hepatocellular tumors in mice of low liver tumor incidence strains. After a helical microorganism was discovered in hepatic parenchyma of diseased mice, we undertook characterization of the organism and investigation of its relationship to the disease process. METHODS: Hepatic histopathology of many strains of mice and rats, as well as guinea pigs and Syrian hamsters, in our research and animal production facilities was reviewed. Steiner's modification of the Warthin-Starry stain and transmission electron microscopy were used to identify bacteria in the liver. We transmitted the hepatitis with liver suspensions from affected mice and by inoculation with bacterial cultures. Bacteria were cultivated on blood agar plates maintained under anaerobic or microaerophilic conditions and characterized morphologically, biochemically, and by 16S rRNA sequence. RESULTS: We report here the isolation of a new species of Helicobacter (provisionally designated Helicobacter hepaticus sp. nov.) that selectively and persistently colonizes the hepatic bile canaliculi of mice (and possibly the intrahepatic biliary system and large bowel), causing a morphologically distinctive pattern of chronic, active hepatitis and associated with a high incidence of hepatocellular neoplasms in infected animals. CONCLUSIONS: The novel Helicobacter is a likely candidate for the etiology of hepatocellular tumors in our mice. The Helicobacter-associated chronic active hepatitis represents a new model to study mechanisms of carcinogenesis by this genus of bacteria. IMPLICATIONS: Adenocarcinoma of the stomach, the second most prevalent of all human malignancies world-wide, is associated with infection at an early age with Helicobacter pylori. Infection leads to several distinctive forms of gastritis, including chronic atrophic gastritis, which is a precursor of adenocarcinoma. H. hepaticus infection in mice constitutes the only other parallel association between a persistent bacterial infection and tumor development known to exist naturally. Study of the H. hepaticus syndrome of chronic active hepatitis and liver tumors in mice may yield insights into the role of H. pylori in human stomach cancer and gastric lymphoma.

Accelerated ultraviolet radiation-induced carcinogenesis in hepatocyte growth factor/scatter factor transgenic mice.

The dramatic rise in incidence of malignant melanoma experienced by populations both within the United States and throughout the world over the last several decades has been attributed to enhanced exposure to the UV spectrum of sunlight radiation. This hypothesis can now be tested using genetically engineered mouse models predisposed to malignant melanoma. Here we use melanoma-prone transgenic mice inappropriately expressing hepatocyte growth factor/scatter factor (HGF/SF) in the skin as an experimental model system to ascertain the consequences of a chronic regimen of suberythemal UV radiation on melanoma genesis. HGF/SF is a multifunctional regulator capable of stimulating growth, motility, invasiveness, and morphogenetic transformation in cells, including melanocytes, expressing its receptor tyrosine kinase Met. HGF/SF transgenic mice demonstrate ectopic interfollicular localization and accumulation of melanocytes within the truncal dermis, epidermis, and junction and if untreated develop primary cutaneous melanoma with a mean onset age of approximately 21 months. Transgenic mice and their wild-type littermates subjected to UV radiation three times weekly using FS40 sunlamps (60% UVB and 40% UVA), with daily UV doses graded from 2.25 to 6.0 kJ/m2, developed skin tumors with a mean onset age of 26 and 37 weeks, respectively (P < 0.001, Kaplan-Meier log rank test). However, the repeated doses of suberythemal UV radiation used in this study failed to accelerate melanoma genesis, instead inducing the development of nonmelanoma tumors that included squamous cell carcinomas, squamous papillomas, and sarcomas. The conspicuous absence of melanocytic tumors occurred despite the immunohistochemical detection of a significant stimulation (P < 0.001) in melanocyte-specific bromodeoxyuridine incorporation in response to only 2 weeks of UV irradiation (total UV dose of 13.5 kJ/m2), resulting in 2.6- and 4.6-fold increases in the number of melanocytes in the dermis and epidermis, respectively. These data indicate that chronic suberythemal UV radiation preferentially favors the development of nonmelanocytic over melanocytic neoplasms in this transgenic animal, consistent with the pathogenesis proposed for sun exposure-associated skin cancer based on retrospective studies in the human population. Our findings suggest that the HGF/SF transgenic mouse will be useful as an experimental model for determining the consequences of exposure to various regimens of UV radiation and for elucidating the mechanisms by which such consequences are realized.

Collaboration between growth factors and diverse chemical carcinogens in hepatocarcinogenesis of transforming growth factor alpha transgenic mice.

Transforming growth factor alpha (TGF-alpha) has been shown to induce liver tumors within 1 year in transgenic male mice in which this potent mitogen is overexpressed. To determine more precisely how TGF-alpha participates in multistep tumorigenesis of the liver, genotoxic (diethylnitrosamine or dimethylnitrosamine) and nongenotoxic (phenobarbital) chemical carcinogens were administered independently to TGF-alpha transgenic mice [line MT42 on a Crl:CD-1(ICR)BR background]. TGF-alpha overexpression dramatically accelerated carcinogen-induced hepatocarcinogenesis in MT42 males but not females. Interestingly, all three chemical agents were found to enhance strongly both hepatic tumor formation and progression in TGF-alpha transgenic male mice. In this study 100%, 90%, and 78% of transgenic males exposed to diethylnitrosamine, dimethylnitrosamine or phenobarbital, respectively, developed tumors between 24 and 32 weeks of age. Moreover, approximately 70% of tumor-bearing transgenic mice from each treatment group had hepatocellular carcinomas; no malignant lesions were found in any carcinogen-treated or untreated nontransgenic mice or in untreated MT42 mice at this age. These results demonstrate that chemical agents as diverse as nitrosamines and phenobarbital act as cocarcinogens with TGF-alpha in the livers of these transgenic mice, indicating that TGF-alpha possesses the unique ability to complement both initiation and promotion in hepatocarcinogenesis. Furthermore, because carcinogen-induced malignant conversion was restricted to transgenic mice, constitutive TGF-alpha overexpression may promote liver tumor progression as well.

Promotion of N-nitrosodiethylamine-initiated hepatocellular tumors and hepatoblastomas by 2,3,7,8-tetrachlorodibenzo-p-dioxin or Aroclor 1254 in C57BL/6, DBA/2, and B6D2F1 mice.

To investigate the hypothesis that tumor promotion by chlorinated aromatic hydrocarbons involves Ah receptor occupation and subsequent induction of cytochromes P-450 1a-1, effects of Aroclor 1254 or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were examined in N-nitrosodiethylamine-initiated mice with different Ah receptor phenotype. Levels of cytochromes P-450 1a and 2b were measured by enzyme assay and Western immunoblots. Males of the C57BL/6, DBA/2, or (C57BL/6 x DBA/2)F1 (hereafter referred to as "B6D2F1") strain were initiated with a single i.p. dose of N-nitrosodiethylamine (90 mg/kg body weight), followed by either multiple doses of TCDD (0.05 micrograms/kg) weekly or Aroclor 1254 chronically in the diet (100 ppm) for 20 weeks, and then no treatment for 24 weeks. Lung tumor incidence or multiplicity was not altered by either of the promoters. Liver tumor incidence was similar among the three strains after N-nitrosodiethylamine alone (14, 21, and 21%, respectively). In DBA/2 mice, TCDD neither induced Cyp 1a nor promoted liver tumors. Aroclor caused an 8-fold induction of hepatic Cyp 2b, which was its maximum at the 12-week time point but did not promote tumors. Inductions of hepatic Cyp 1a by TCDD and 1a and 2b by Aroclor were similar in C57BL/6 and B6D2F1 mice, but tumor promotion responses were quite different. Dietary Aroclor significantly promoted liver tumors in C57BL/6 mice (59 versus 14%) but not in B6D2F1 mice (24 versus 21%). Repeated TCDD promoted only in B6D2F1 mice (52 versus 21%) and not in C57BL/6 mice (19 versus 14%). Thus, whereas these data confirm that a functional Ah receptor is required for liver tumor promotion, the degree of activation as measured by induction of Cyp 1a is not directly related to the degree of tumor-promoting capability. Other genetic factors must play a role in mediating the final tumor outcome.

2-difluoromethylornithine and dehydroepiandrosterone inhibit mammary tumor progression but not mammary or prostate tumor initiation in C3(1)/SV40 T/t-antigen transgenic mice.

Female transgenic mice that express SV40 T/t antigens under the regulatory control of the rat C3(1) gene spontaneously develop multifocal mammary lesions that predictably evolve into invasive, hormone-independent carcinomas, whereas male mice are prone to develop prostate cancer. Chemopreventive agents were administered to female C3(1)/SV40 large T-antigen mice from 7 to 19 weeks of age, during which time the mammary lesions developed and progressed to invasive carcinomas. No significant differences in the numbers of preinvasive mammary intraepithelial neoplasia lesions (histologically similar to human ductal carcinoma in situ) were observed after 2 or 8 weeks of treatment between mice receiving either vehicle alone, dehydroepiandrosterone (DHEA), or 2-difluoromethylornithine (DFMO). However, a dose-response reduction in invasive carcinoma growth was observed for both DFMO, an inhibitor of ornithine decarboxylase, and DHEA, the primary steroid precursor to both androgens and estrogens in primates. Despite unaltered expression of the transgene, tumor incidence was reduced approximately 20% by DFMO (8000 mg/kg) and 30% by DHEA (4000 mg/kg; P < 0.05). Tumor multiplicity was reduced by approximately 50% by both DFMO and DHEA (P < 0.05). DFMO had a dose-dependent effect on total tumor burden, which was reduced by 25% at low doses (4000 mg/kg) and 70% at high doses (8000 mg/kg). DHEA reduced tumor burden by 50% and 66% at low (2000 mg/kg) and high (4000 mg/kg) doses, respectively. Interestingly, despite its inhibitory effects on tumor development, DHEA caused a dose-dependent increase of serum estradiol levels that we have previously shown to increase mammary tumor formation in this model. No effect on the development of the prostate cancer precursor lesions (prostate intraepithelial neoplasia) was observed when mice were treated with DHEA, DFMO, tocopherol acetate, selenomethionine, or 9-cis-retinoic acid, although the effects on late-stage prostate cancer development were not determined. These results demonstrate that despite the expression of the highly transforming C3(1)/SV40 large T-antigen transgene, this transgenic model can be used to study the effects of chemopreventive agents on mammary cancer progression. The tumor-inhibitory effects of DHEA and DFMO on mammary cancer growth appear to occur after the development of preinvasive lesions, suggesting that these agents inhibit tumor progression but not initiation.

Chemopreventive activity of tamoxifen, N-(4-hydroxyphenyl)retinamide, and the vitamin D analogue Ro24-5531 for androgen-promoted carcinomas of the rat seminal vesicle and prostate.

We evaluated the ability of dietary N-(4-hydroxyphenyl)retinamide; 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531); and tamoxifen to inhibit the development of androgen-promoted carcinomas of the accessory sex organs of male Lobund-Wistar rats. Invasive carcinomas of the seminal vesicle (SV) and anterior prostate (AP) were induced in Lobund-Wistar rats with three different combinations of initiator [N-nitroso-N-methylurea (NMU)] and promoter [testosterone propionate (TP)]: (a) high-dose NMU (30 mg/kg) + high-dose TP (20 mg via implant every 2 months); (b) high-dose NMU + low-dose TP (10 mg implanted every 2 months); or (c) low-dose NMU (15 mg/kg) + low-dose TP. During the period of TP administration, rats were fed a diet supplemented with either N-(4-hydroxyphenyl)retinamide (1 or 2 mmol/kg diet), Ro24-5531 (1.25 or 2.5 nmol/kg diet), tamoxifen (0.5 or 5 mg/kg diet), or vehicle alone. After sacrifice at 8.5 or 11 months, the prostate-seminal vesicle complex from each rat was processed in toto and histologically staged as to the extent of tumor involvement. In animals given low-dose TP, all three agents were significantly effective at reducing the incidence of invasive carcinomas of the SV and, to a lesser degree, the AP. Of the three agents, tamoxifen given in high dose (5 mg/kg) had the strongest activity, reducing the occurrence of invasive SV carcinomas from 72-83% in controls to 6% (P = 0.0001) and the occurrence of invasive AP carcinomas from 50-72% to 18-22% (P < 0.05).

The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma.

The 5' flanking region of the C3(1) component of the rat prostate steroid binding protein (PSBP) has been used to successfully target the expression of the SV40 large T-antigen (Tag) to the epithelium of both the mammary and prostate glands resulting in models of mammary and prostate cancers which histologically resemble the human diseases. Atypia of the mammary ductal epithelium develops at about 8 weeks of age, progressing to mammary intraepithelial neoplasia (resembling human ductal carcinoma in situ [DCIS]) at about 12 weeks of age with the development of invasive carcinomas at about 16 weeks of age in 100% of female mice. The carcinomas share features to what has been classified in human breast cancer as infiltrating ductal carcinomas. All FVB/N female mice carrying the transgene develop mammary cancer with about a 15% incidence of lung metastases. Approximately 10% of older male mice develop anaplastic mammary carcinomas. Unlike many other transgenic models in which hormones and pregnancy are used to induce a mammary phenotype, C3(1)/Tag mice develop mammary tumors in the mammary epithelium of virgin animals without hormone supplementation or pregnancy. Although mammary tumor development appears hormone-responsive at early stages, invasive carcinomas are hormone-independent, which corresponds to the loss of estrogen receptor-alpha expression during tumor progression. Molecular and biologic factors related to mammary tumor progression can be studied in this model since lesions evolve over a predictable time course. Genomic alterations have been identified during tumor progression, including an amplification of the distal portion of chromosome 6 containing ki-ras and loss of heterozygosity (LOH) in other chromosomal regions. We have demonstrated that stage specific alterations in the expression of genes which are critical regulators of the cell cycle and apoptosis are functionally important in vivo. C3(1)/Tag mice appear useful for testing particular therapies since growth of the mammary tumors can be reduced using chemopreventive agents, cytokines, and an anti-angiogenesis agent.

The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting.

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

Lsh, a SNF2 family member, is required for normal murine development.

Lsh is a member of the SNF2 family of chromatin remodelers, that regulate diverse biological processes such as replication, repair and transcription. Although expression of Lsh is highly tissue specific in adult animals, Lsh mRNA is detectable in multiple tissues during embryogenesis. In order to determine the physiologic role of Lsh during murine development and to assess its unique function in adult mice, we performed targeted deletion of the Lsh gene using homologous recombination in murine embryonic stem cells. Lsh-/- embryos occurred with the expected Mendelian frequency after implantation and during embryogenesis. However, Lsh-/- mice died within a few hours after birth. Furthermore, newborn mice were 22% lower in weight in comparison with their littermates and showed renal lesions. Thus Lsh is a non-redundant member of the SNF2 family and is essential for normal murine development and survival.

Age-related alterations in 32P-postlabeled DNA adducts in livers of mice infected with the tumorigenic bacterial pathogen, Helicobacter hepaticus.

Helicobacter hepaticus causes chronic active hepatitis and liver tumors in mice, with associated increase in reactive oxygen species. Indigenous (I)-compounds are bulky DNA adducts present at low levels and detected by 32P-post-labeling. Some may be caused by reactive oxygen species; others occur normally and decrease during liver tumorigenesis. The identity of most is unknown. We investigated whether mouse liver infection by H. hepaticus and resulting progression of hepatic lesions would be associated with qualitative or quantitative changes in I-compounds. Mice were 3, 6, 9, and 12 months of age; liver disease ranged from minimal through marked. In control A/J mice, up to 20 I-compounds were detected, and the total level of these did not change with age, whereas 11 individual I-compounds showed marked age-related differences. These appeared to be coordinately regulated, as the total of these 11 adducts was constant at 6-12 months. In A/JNCr mice naturally infected with H. hepaticus, up to 12 hepatic I-compounds were found. Total levels varied markedly with age and were high at 6 and 12 months. Neither total adduct levels, nor the amount of any individual adduct, correlated positively with severity of hepatic lesions; in some cases, highest levels were found in livers with least disease. Thus, liver infection and tumorigenesis by H. hepaticus was not associated with an increase in any 32P-postlabeled DNA adduct. Marked, and distinct, age-related changes in total or individual adducts in control and infected mice suggest a role in the physiological alterations of aging and in host response to infection.

Renal artery occlusion and infarction with Sotradecol.

Sodium tetradecyl sulfate 3% (Sotradecol) was perfused into a proximally obstructed renal artery in ten random-source adult dogs. In all animals, the intra-arterial injections of Sotradecol produced immediate thrombosis of the renal arteries. Angiographic studies, lasting up to ten weeks, on eight dogs demonstrated persistent occlusion. In one dog, unintentional thrombosis occurred in the opposite renal artery due to reflux of Sotradecol. Histopathologic studies showed total obliteration of the renal arteries through organized thrombosis, intimal sclerosis, and luminal collapse. In all dogs, the kidneys were not visible, and diffuse renal infarction was confirmed by histologic studies of five dogs.

Cyclooxygenase-2, P-glycoprotein-170 and drug resistance; is chemoprevention against multidrug resistance possible?

BACKGROUND: It is generally accepted that P-glycoprotein 170 (MDR1/Pgp170) expression in breast tumors results in poor response to chemotherapy due to its ability to export chemotherapeutic agents. Studies indicate that the use of non-steroidal anti-inflammatory drugs (NSAIDs) may enhance the anti-tumor activity of cancer chemotherapeutic agents and reduce the risk of many cancers. The best known function of NSAIDs is to block the enzyme cyclooxygenase (Cox), the rate limiting enzyme in the conversion of arachidonic acid to prostaglandins. In this study we investigated whether expression of the inducible isoform of Cox (Cox-2) is linked with the multidrug resistance phenotype in breast cancer. METHODS: Expression of Cox-2 and MDR1/Pgp170 was investigated in tumor specimens along with normal epithelium in breast cancer patients using immunohistochemisrty. Expression of Cox-2, MDR1/Pgp170, Protein Kinase C (PKC), and Activator Protein 1 (AP1) were investigated in a series of increasingly resistant human MCF-7 breast cancer cells compared to wild type using immunohistochemistry, Western blots, Northern blots, RT-PCR, and Southern blots. RESULTS: Immunohistochemical analyses of human breast tumor specimens revealed a strong correlation between expression of Cox-2 and MDR1/Pgp170. In drug resistant cell lines that over-express MDR1/Pgp170 there was also significant up-regulation of Cox-2 expression. In addition, PKC and AP1 subunits c-Jun and c-Fos were also upregulated. We hypothesized that increased prostaglandin production by Cox-2 induces PKC and the expression of transcriptional factor c-Jun, which in turn, induces the expression of MDR1/Pgp170. CONCLUSION: We propose that pretreatment with selective Cox-2 inhibitors may be useful in the prevention of multidrug resistance in response to cancer chemotherapy and should be further evaluated.

High incidence of neoplasms in female NZB/NZW mice treated with pulse doses of cyclophosphamide.

The immunosuppressive properties of cyclophosphamide prevent formation of anti-DNA antibodies and prolong lifespans in autoimmune NZB/NZW mice, an animal model of systemic lupus erythematosus. In the current study, NZB/NZW mice were treated with weekly doses of cyclophosphamide to determine if intermittent pulses of the drug were effective therapy. Life-long treatment with cyclophosphamide, 56 mg/kg/week, was started at the mean age of 6 weeks; results were compared with saline-injected control mice. Pulse therapy with cyclophosphamide suppressed anti-DNA antibody levels, prevented severe glomerulonephritis and prolonged longevity. Seventeen of 19 treated mice developed neoplasms; 7 of these immunosuppressed animals had 2 to 4 separate neoplasms. Examination of earlier studies in this laboratory in which NZB/NZW mice were treated each day with cyclophosphamide showed that daily and weekly therapeutic regimens had similar immunosuppressive and oncogenic effects.

Chronic toxic and carcinogenic effects of oral cadmium in the Noble (NBL/Cr) rat: induction of neoplastic and proliferative lesions of the adrenal, kidney, prostate, and testes.

Based on the occurrence of pulmonary cancers in exposed populations, cadmium is classified as a human carcinogen. More controversial target sites for cadmium in humans include the prostate and kidney, where some studies have shown a link between cadmium and cancer. In Wistar rats cadmium induces tumors in the ventral prostate. The relevance of such lesions to humans is debated since the rat ventral lobe, unlike the dorsolateral lobe, has no embryological homolog in the human prostate. Cadmium has not been linked with renal tumors in rodents but is a potent nephrotoxin. In this work we studied the effects of oral cadmium in the Noble (NBL/Cr) rat with particular attention to proliferative lesions of the prostate and kidneys. Cadmium (as CdCl2) was given ad libitum throughout the study in the drinking water at doses of 0, 25, 50, 100, and 200 ppm Cd to groups (initial n = 30) of male rats, which were observed for up to 102 wk. At the lower doses of cadmium (< or =50 ppm) a clear dose-related increase in total proliferative lesions of the prostate (ventral and dorsolateral lesions combined) occurred (0 ppm = 21% incidence, 25 ppm = 46%, 50 ppm = 50%; trend p < .03). These lesions were described as intraepithelial hyperplasia with occasional areas of atypical epithelial cells without stromal invasion. The lesions occurred primarily in the dorsolateral prostate with cadmium exposure and most frequently showed three or more foci within each specimen. At higher doses, prostatic proliferative lesions declined to control levels. The loss of prostatic response at the higher doses was likely due to diminished testicular function secondary to cadmium treatment. This was reflected in lesions indicative of testicular hypofunction at the highest cadmium dose, namely, interstitial cell hyperplasia, and a strong correlation between cadmium dose and total proliferative lesions of the testes (hyperplasias and tumors combined). Renal tumors (mainly mesenchymal and pelvic transitional cell), although few in number, showed a positive correlation with cadmium dose, as did pelvic transitional epithelial hyperplasia. Renal lesions were not associated with any cadmium-induced changes in age-related chronic nephropathy. The incidence of pheochromocytomas of the adrenal was increased by cadmium but only at the 50 ppm dose. Inflammatory lesions of the liver and spleen were common at higher doses and showed strong trends based on dose. These results indicate that oral cadmium can induce proliferative lesions in the prostate and kidney of the Noble rat. The finding of proliferative lesions of dorsolateral prostate in rats has presumed relevance to human prostate cancers.