Kenji Kamino

Testing the importance of p27 degradation by the SCFskp2 pathway in murine models of lung and colon cancer

Decreased expression of the CDK inhibitor p27kip1 in human tumors directly correlates with increased resistance to chemotherapies, increased rates of metastasis, and an overall increased rate of patient mortality. It is thought that decreased p27 expression in tumors is caused by increased proteasomal turnover, in particular activation of the pathway governed by the SCFskp2 E3 ubiquitin protein ligase. We have directly tested the importance of the SCFskp-mediated degradation of p27 in tumorigenesis by analyzing the tumor susceptibility of mice that express a form of p27 that cannot be ubiquitinated and degraded by this pathway (p27T187A). In mouse models of both lung and colon cancer down-regulation of p27 promotes tumorigenesis. However, we found that preventing p27 degradation by the SCFskp2 pathway had no impact on tumor incidence or overall survival in either tumor model. Our study unveiled a previously unrecognized role for the control of p27 mRNA abundance in the development of non-small cell lung cancers. In the colon cancer model, the frequency of intestinal adenomas was similarly unaffected by the p27T187A mutation, but, unexpectedly, we found that it inhibited progression of intestinal adenomas to carcinomas. These studies may guide the choice of clinical settings in which pharmacologic inhibitors of the Skp2 pathway might be of therapeutic value.

Significant Differences in the Effects of Magnetic Field Exposure on 7,12-Dimethylbenz(a)anthracene-Induced Mammary Carcinogenesis in Two Substrains of Sprague-Dawley Rats

We have shown previously (S. Thun-Battersby et al., Cancer Res., 59: 3627–3633, 1999) that power-line frequency (50-Hz) magnetic fields (MFs) at μT-flux densities enhance mammary gland tumor development and growth in the 7,12-dimethylbenz(a)anthracene (DMBA) model of breast cancer in female Sprague-Dawley (SD) rats. We also demonstrated that MF exposure results in an enhanced proliferative activity of the mammary epithelium of SD rats (M. Fedrowitz et al., Cancer Res., 62: 1356–1363, 2002), which is a likely explanation for the cocarcinogenic or tumor-promoting effects of MF exposure in the DMBA model. However, in contrast with our data, in a similar study conducted by Battelle in the United States, no evidence for a cocarcinogenic or tumor-promoting effect of MF exposure was found in the DMBA model in SD rats (L. E. Anderson et al., Carcinogenesis, 20: 1615–1620, 1999). Probably the most important difference between our and the Battelle studies was the use of different substrains of SD rats; the United States rats were much more susceptible to DMBA than the rats used in our studies. This prompted us to compare different substrains of SD outbred rats in our laboratory in respect to MF effects on cell proliferation in the mammary gland, susceptibility to DMBA-induced mammary cancer, and MF effects on mammary tumor development and growth in the DMBA model. The SD substrain (termed “SD1”) used in all of our previous studies was considered MF-sensitive and used for comparison with another substrain (“SD2”) obtained from the same breeder. In contrast with SD1 rats, no enhanced cell proliferation was determined after MF exposure in SD2 rats. MF exposure significantly increased mammary tumor development and growth in SD1 but not SD2 rats. These data indicate that the genetic background plays a pivotal role in effects of MF exposure. Different strains or substrains of rats may serve to evaluate the genetic factors underlying sensitivity to cocarcinogenic or tumor-promoting effects of MF exposure.

Results of current intraperitoneal carcinogenicity studies with mineral and vitreous fibres

The study includes some 50 groups of male or female Wistar rats tested in three series. Except for one untreated group and 3 vehicle control groups, the animals were injected intraperitoneally (i.p.) once or repeatedly with dust suspensions and then examined, after lifetime observation up to 30 months, for tumours in the abdominal cavity. 1 granular dust (silicon carbide), 2 asbestos dusts (crocidolite, tremolite) and 11 vitreous fibre dust samples were administered. 5 of the vitreous fibre types were fine fibre fractions from 4 commercial insulation wools and 1 experimental wool, the others were prepared by milling glass microfibres, which have, per se, a small diameter range. The dosage per rat differed over a wide range in accordance with experience from earlier studies. The lowest dose was 0.04 x 10(9) crocidolite fibres in 0.5 mg dust, and the highest amounted to 20 x 10(9) glass fibres in 1000 mg divided into 40 weekly injections. Two mesotheliomas were found in a total of 395 rats treated with saline or granular silicon carbide (250 or 1000 mg). Eleven fibre dusts produced dose-dependent mesotheliomas at rates of up to 97 %, but the calculated fibre number > 5 micrometers in length required for inducing a 25 % tumour risk differed between the fibre samples tested in the relation of 1 to about 1000. UICC-like crocidolite heads the ranking order; the glass fibre B-01, which possesses a low durability in the body, ends it together with a rather thin sample of glass fibre type B-09. The stone fibre MMVF-21 takes a high place in the ranking order, similar to the tremolite sample. The results correspond to those of earlier i.p. tests.

Subcutaneous soft tissue tumours at the site of implanted microchips in mice

An experiment using 4279 CBA/J mice of two generations was carried out to investigate the influence of parental preconceptual exposure to X-ray radiation or to chemical carcinogens. Microchips were implanted subcutaneously in the dorsolateral back for unique identification of each animal. The animals were kept for lifespan under standard laboratory conditions. In 36 mice a circumscribed neoplasm occurred in the area of the implanted microchip. Females were significantly more frequently affected than male mice. An influence of age or different treatment on the s.c. tumour incidence in two mice generations could not be observed. Macroscopically, firm, pale white nodules up to 25 mm in diameter with the microchip in its center were found. Microscopically, soft tissue tumours such as fibrosarcoma and malignant fibrous histiocytoma were detected.

Treatment of lupus-prone NZB/NZW F1 mice with recombinant soluble Fcγ receptor II (CD32)

Objectives: Systemic lupus erythematosus (SLE) is a classical autoimmune disorder characterised by the production of IgG autoantibodies against double-stranded DNA (dsDNA). Activation of FcγR-bearing effector cells by immune complexes (ICs) is a key event in SLE pathogenesis as lupus-prone NZB/NZW F1 hybrids lacking activating Fcγ receptors (FcγR) are protected against inflammatory kidney damage despite glomerular deposition of ICs. Moreover, soluble FcγRs inhibit IC-caused Arthus reaction in vivo. Therefore, recombinant human soluble FcγRII (CD32) was evaluated as a novel therapeutic strategy in lupus-like disease in NZB/NZW F1 hybrids. Methods: Binding of husCD32 to murine IgG was studied in vitro by binding to IgG-coated erythrocytes and inhibition of phagocytosis of IgG-opsonised murine erythrocytes. In order to examine therapeutic impact of husCD32 in vivo, female NZB/NZW F1 mice were treated either from week 16 to 20 (“prophylactic”, 150 μg/week husCD32) or continuously from week 24 (“therapeutic”; 100 μg/week husCD32) by subcutaneous injections. Controls received buffered saline. Results: In vitro investigations of husCD32 revealed binding to murine erythrocytes coated with murine IgG. Moreover, husCD32 substantially diminished phagocytosis of murine IgG-opsonised murine red blood cells by peritoneal macrophages indicating disruption of IgG–FcγR interaction. There was a therapeutic efficacy of husCD32 to attenuate lupus pathology indicated by significantly delayed onset of proteinuria and weight loss, reduced histopathological findings, delayed development of anaemia and improved survival by prophylactical application. Therapeutic treatment did not reverse nephritis but significantly prolonged survival despite apparent kidney damage. B cell count, concentration of IgG anti-dsDNA autoantibodies and deposition of glomerular ICs was not significantly affected by the application of husCD32. Conclusions: The results demonstrate binding properties of husCD32 to ICs in vitro and as a proof-of-principle therapeutic efficacy in inhibiting chronic murine lupus pathology in vivo.

Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice.

Ki‐ras and p53 genes are involved in human lung carcinogenesis; however, the role of these genes in experimental lung tumors is not well known. In our study, the CBA/J mouse strain was used to investigate the presence of Ki‐ras and p53 alterations in lung carcinogenesis of spontaneous tumors and tumors induced with high and low doses of urethane (ethyl carbamate). To study the presence of these alterations in the early stages of lung carcinogenesis and in very small lung tumors, restriction fragment length polymorphism and single‐strand conformation polymorphism analyses were performed on polymerase chain reaction–amplified DNA from microdissected tumoral and normal lung samples. Ki‐ras gene mutations in codons 12 and 61 were detected in all types of lung lesions, even in small and preneoplastic lesions, and their incidence increased with progression from lung hyperplasias (18%) to adenomas (75%) and to carcinomas (80%). Urethane exposure, in both high and low doses, increased the incidence of Ki‐ras mutations in lung tumors, especially in adenomas. The presence of Ki‐ras gene mutations in very small urethane‐induced lung tumors and the absence of hyperplasias among the treated‐group lesions may indicate that urethane accelerates tumoral progression. No p53 mutations were detected in exons 5–8 in any of the epithelium‐derived lung tumors. Only one p53 mutation in exon 5 was found in a spontaneous lymphoma. Therefore, p53 mutations do not seem to cooperate with Ki‐ras gene mutations or represent an alternative molecular pathway in murine carcinogenesis. Mol. Carcinog. 21:251–260, 1998.

Strain-specific mammary proliferative lesion development following lifetime oral administration of ochratoxin A in DA and Lewis rats

OTA, a potent nephrotoxin in several species, is a renal carcinogen in animals and is implicated in the etiology of BEN. The NTP classified OTA as having clear evidence of carcinogenic activity, based on uncommon tubular adenomas and tubular cell carcinomas of the kidney and multiple fibroadenomas of the mammary gland, seen in the rat. As shown previously (Castegnaro et al., Int J Cancer 1998;77:70–5), induction of renal tumors by OTA is sex‐ and strain‐specific in DA and Lewis rats, with DA males being most responsive and DA females being resistant; however, that report was confined to the kidney and urinary tract. To obtain OTA‐induced tumorigenic information in rats, we administered OTA (0.4 mg/kg) by oral gavage to both DA and Lewis rats for their lifetimes and extended the investigation to complete histopathology of all tissues and organs. We also observed the characteristic renal tumor that is highly strain‐ and sex‐specific, and there were increased incidences of proliferative mammary lesions in Lewis rats but not in DA rats, indicating that these were also strain‐specific. In view of the NTP report of OTA treatment‐related mammary fibroadenoma in F344 rats, we observed increased mammary proliferative lesions in Lewis rats but not in DA rats. Our results suggest that OTA may play some role in mammary tumor development in some rat strains.

Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus

The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37–75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O6-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.

Insertional Mutagenesis by Replication‐Deficient Retroviral Vectors Encoding the Large T Oncogene

Abstract:  Insertion sites of replication‐deficient retroviral vectors may trigger clonal dominance of hematopoietic cells in vivo. Here, we tested whether this would also be the case when using vectors that express powerful oncogenes, such as the large tumor antigen (TAg) of simian virus 40. TAg inactivates the tumor‐suppressor proteins p53 and Rb by virtue of a chaperone‐like activity. Primary hematopoietic stem/progenitor cells transduced with retroviral vectors encoding TAg‐induced histiocytic sarcoma (HS) or myeloid leukemia (ML) in transplanted mice (average survival of 21 weeks). Retrovirally introducing TAg into pretransformed 32D cells generated a monocytic leukemia, with faster kinetics (∼8 weeks). Leukemic clones showed retroviral insertions in genes contributing to all known TAg cooperation pathways, acting mitogenic and/or modulating apoptosis (such as BclX, Crk, Pim2, Csfr1/Pdgfrb, Osm/Lif, Axl, Fli, Sema4b, Sox4). 32D‐derived monocytic leukemias showed hits in Pim2 and Max proto‐oncogenes, or the chaperone Hspa4, plus additional signaling genes. Vector‐mediated insertional mutagenesis thus revealed a broad spectrum of potential TAg complementation genes. These findings have important implications for the use of retroviral transgenesis in cancer research, and the expression of signaling genes in somatic gene therapy.

Spectrum and age-related incidence of spontaneous tumours in a colony of Han:AURA hamsters

One-hundred-and-forty-four male and 184 female untreated Syrian golden hamsters (strain Han:AURA) were kept for life under standard laboratory conditions. They were examined with regard to spontaneously occurring tumours in relation to their survival periods. The mean survival rate of the males was 106 +/- 26 weeks and that of the females 97 +/- 20 weeks. Tumours were found in 71% of males and 67% of females. Adenomas and carcinomas of the adrenal glands were the most frequently observed tumours in both sexes (male: 66%; female: 38%) and in the early stages of life. Malignant lymphoma (8%), adenomas and carcinomas of pancreatic islet-cells (8%) and papillomatous benign and malignant squamous cell tumours of the forestomach (7%) showed relatively high incidences in males, whilst in females, leiomyoma (10%) and endometrial adenocarcinoma (7%) of the uterus and adenomas and carcinomas in the pars distalis of the pituitary gland (9%) occurred frequently.