Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas.

The gene defect for hereditary papillary renal carcinoma (HPRC) has recently been mapped to chromosome 7q, and germline mutations of MET (also known as c-met) at 7q31 have been detected in patients with HPRC (ref. 2). Tumours from these patients commonly show trisomy of chromosome 7 when analysed by cytogenetic studies and comparative genomic hybridization (CGH). However, the relationship between trisomy 7 and MET germline mutations is not clear. We studied 16 renal tumours from two patients with documented germline mutations in exon 16 of MET. Fluorescent in situ hybridization (FISH) analysis showed trisomy 7 in all tumours. To determine whether the chromosome bearing the mutant or wild-type MET gene was duplicated, we performed duplex PCR and phosphoimage densitometry using polymorphic microsatellite markers D7S1801 and D7S1822, which were linked to the disease gene locus, and D1S1646 as an internal control. We determined the parental origin of chromosome alleles by genotyping parental DNA. In all 16 tumours there was an increased signal intensity (2:1 ratio) of the microsatellite allele from the chromosome bearing the mutant MET compared with the allele from the chromosome bearing the wild-type MET. Our study demonstrates a non-random duplication of the chromosome bearing the mutated MET in HPRC and implicates this event in tumorigenesis.

Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.

Hereditary papillary renal carcinoma (HPRC) is a recently recognized form of inherited kidney cancer characterized by a predisposition to develop multiple, bilateral papillary renal tumours. The pattern of inheritance of HPRC is consistent with autosomal dominant transmission with reduced penetrance. HPRC is histologically and genetically distinct from two other causes of inherited renal carcinoma, von Hippel-Lindau disease (VHL) and the chromosome translocation (3;8). Malignant papillary renal carcinomas are characterized by trisomy of chromosomes 7, 16 and 17, and in men, by loss of the Y chromosome. Inherited and sporadic clear cell renal carcinomas are characterized by inactivation of both copies of the VHL gene by mutation, and/or by hypermethylation. We found that the HPRC gene was located at chromosome 7q31.1-34 in a 27-centimorgan (cM) interval between D7S496 and D7S1837. We identified missense mutations located in the tyrosine kinase domain of the MET gene in the germline of affected members of HPRC families and in a subset of sporadic papillary renal carcinomas. Three mutations in the MET gene are located in codons that are homologous to those in c-kit and RET, proto-oncogenes that are targets of naturally-occurring mutations. The results suggest that missense mutations located in the MET proto-oncogene lead to constitutive activation of the MET protein and papillary renal carcinomas.

Treatment by limited surgery and specific immunization of guinea pigs with stage II experimental cancer.

The malignant disease produced in guinea pigs by intradermal inoculation of line-10 was allowed to progress to stage II, at which time the dermal tumor and the first draining lymph node were grossly evident. At that stage, the external appearance of the next draining lymph node was normal, but it contained tumor cells. Limited surgery consisting of excision of the dermal tumor and first draining lymph node was not curative; palpable metastases developed in the second and other draining lymph nodes, and at autopsy, some animals were found to have gross, visible lung metastases. Immunization of guinea pigs with a mixture of irradiated syngeneic tumor cells plus mycobacterial cell walls in an oil-in-water emulsion eradicated tumor cells remaining in lymph nodes after limited surgery for stage II experimental cancer and prevented progression of the disease to stage III. Tumor intravenously implanted in the lungs of animals after limited surgery for stage II disease was also eliminated by immunization.

Immunotherapy of cancer: regression of tumors after intralesional injection of living Mycobacterium bovis.

Injection of living Mycobacterium bovis (strain BCG) into established intradermal tumors caused tumor regression and prevented the development of metastases.

Tumor immunity produced by the intradermal inoculation of living tumor cells and living Mycobacterium bovis (strain BCG).

The intradermal inoculation of mixtures containing living tumor cells and living Mycobacterium bovis (strain BCG) into unimmunized syngeneic guinea pigs results in an inflammatory reaction to the BCG, and there is no progressive tumor growth. In the absence of BCG the tumor grows progressively, metastasizes, and kills the animal. By conventional methods, it has not been possible to immunize syngeneic guinea pigs to the tumor used. Guinea pigs that receive mixtures of BCG and tumor cells, however, develop specific systemic tumor immunity as measured by delayed cutaneous hypersensitivity and by suppression of tumor growth.

Tumor immunity: tumor suppression in vivo initiated by soluble products of specifically stimulated lymphocytes.

Supernatant fluids of specifically stimulated lymphocyte cultures were purified. Fractions containing migration inhibition factor when injected intra-dermally into strain-2 guinea pigs produced a reaction similar in appearance to delayed cutaneous hypersensitivity. There was an accumulation of mononuclear cells at the injection sites and the growth of syngeneic tumor grafts at the sites was suppressed.

Immunotherapy of cancer: an experimental model in syngeneic guinea pigs.

Successful treatment of a solid tumor was accomplished by repeated intradermal injection of living tumor cells.

Tumor-specific antigens detected by inhibition of macrophage migration.

Tumor-specific antigens of a guinea pig hepatoma induced by diethylnitrosamine were detected by the inhibition of migration of specifically sensitized macrophages from capillary tubes, and by the local passive transfer of delayed skin hypersensitivity and the suppression of growth of intradermally same injected tumor.

A transforming ras gene in tumorigenic guinea pig cell lines initiated by diverse chemical carcinogens.

Fetal guinea pig cells were transformed by treatment with four different chemical carcinogens including nitroso compounds and polycyclic hydrocarbons. As a consequence of this treatment, oncogenes capable of transforming NIH/3T3 cells became activated in each of five independently established clonal guinea pig cell lines. Molecular characterization of representative NIH/3T3 transformants revealed that the same oncogene was present in each of the cell lines tested. Moreover, detection of this transforming gene paralleled the acquisition of tumorigenic properties by these neoplastic cells.

The Drosophila homolog of the human tumor suppressor gene BHD interacts with the JAK-STAT and Dpp signaling pathways in regulating male germline stem cell maintenance.

Birt-Hogg-Dubé syndrome (BHD) is a rare, inherited genodermatosis characterized by hair follicle hamartomas, kidney tumors and spontaneous pneumothorax. The BHD locus was mapped to chromosome 17p11.2 by linkage analysis, and germline mutations in a novel gene (BHD) were identified in a panel of BHD families. Using RNA interference to decrease expression of the Drosophila BHD homolog (DBHD), we have demonstrated that DBHD is required for male germline stem cell (GSC) maintenance in the fly testis. Reduction of DBHD gene activity suppresses the GSC overproliferation phenotype associated with overexpression of either unpaired (upd) or decapentaplegic (dpp). Further genetic interaction experiments suggest that DBHD regulates GSC maintenance downstream or in parallel of the JAK/STAT and Dpp signal-transduction pathways. These findings suggest that the BHD protein may regulate tumorigenesis through modulating stem cells in human.

Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein.

The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17-50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157-171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.

Oncogenic mutants of RON and MET receptor tyrosine kinases cause activation of the beta-catenin pathway.

beta-Catenin is an oncogenic protein involved in regulation of cell-cell adhesion and gene expression. Accumulation of cellular beta-catenin occurs in many types of human cancers. Four mechanisms are known to cause increases in beta-catenin: mutations of beta-catenin, adenomatous polyposis coli, or axin genes and activation of Wnt signaling. We report a new cause of beta-catenin accumulation involving oncogenic mutants of RON and MET receptor tyrosine kinases (RTKs). Cells transfected with oncogenic RON or MET were characterized by beta-catenin tyrosine phosphorylation and accumulation; constitutive activation of a Tcf transcriptional factor; and increased levels of beta-catenin/Tcf target oncogene proteins c-myc and cyclin D1. Interference with the beta-catenin pathway reduced the transforming potential of mutated RON and MET. Activation of beta-catenin by oncogenic RON and MET constitutes a new pathway, which might lead to cell transformation by these and other mutant growth factor RTKs.

Protective function of von Hippel-Lindau protein against impaired protein processing in renal carcinoma cells.

The absence of functional von Hippel-Lindau (VHL) tumor suppressor gene leads to the development of neoplasias characteristic of VHL disease, including renal cell carcinoma (RCC). Here, we compared the sensitivity of RCC cells lacking VHL gene function with that of RCC cells expressing the wild-type VHL gene (wtVHL) after exposure to various stresses. While the response to most treatments was not affected by the VHL gene status, glucose deprivation was found to be much more cytotoxic for RCC cells lacking VHL gene function than for wtVHL-expressing cells. The heightened sensitivity of VHL-deficient cells was not attributed to dissimilar energy requirements or to differences in glucose uptake, but more likely reflects a lesser ability of VHL-deficient cells to handle abnormally processed proteins arising from impaired glycosylation. In support of this hypothesis, other treatments which act through different mechanisms to interfere with protein processing (i.e., tunicamycin, brefeldin A, and azetidine) were also found to be much more toxic for VHL-deficient cells. Furthermore, ubiquitination of cellular proteins was elevated in VHL-deficient cells, particularly after glucose deprivation, supporting a role for the VHL gene in ubiquitin-mediated proteolysis. Accordingly, the rate of elimination of abnormal proteins was lower in cells lacking a functional VHL gene than in wtVHL-expressing cells. Thus, pVHL appears to participate in the elimination of misprocessed proteins, such as those arising in the cell due to the unavailability of glucose or to other stresses.

Fumarate hydratase enzyme activity in lymphoblastoid cells and fibroblasts of individuals in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. OBJECTIVE: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. METHODS: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. RESULTS: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p<0.05). The enzyme activity in lymphoblastoid cell lines from three individuals with mutations in R190 was not significantly different from individuals with other missense mutations. The cytosolic and mitochondrial FH activity of cell lines from individuals with HLRCC was reduced compared with those from control cell lines (p<0.05). There was no significant difference in enzyme activity between control cell lines (n = 4) and cell lines from affected individuals with other hereditary renal cancer syndromes (n = 22). CONCLUSIONS: FH enzyme activity testing provides a useful diagnostic method for confirmation of clinical diagnosis and screening of at-risk family members.

Novel mutations in FH and expansion of the spectrum of phenotypes expressed in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC; OMIM 605839) is the predisposition to develop smooth muscle tumours of the skin and uterus and/or renal cancer and is associated with mutations in the fumarate hydratase gene (FH). Here we characterise the clinical and genetic features of 21 new families and present the first report of two African-American families with HLRCC. METHODS: Using direct sequencing analysis we identified FH germline mutations in 100% (21/21) of new families with HLRCC. RESULTS: We identified 14 germline FH mutations (10 missense, one insertion, two nonsense, and one splice site) located along the entire length of the coding region. Nine of these were novel, with six missense (L89S, R117G, R190C, A342D, S376P, Q396P), one nonsense (S102X), one insertion (111insA), and one splice site (138+1G>C) mutation. Four unrelated families had the R58X mutation and five unrelated families the R190H mutation. Of families with HLRCC, 62% (13/21) had renal cancer and 76% (16/21) cutaneous leiomyomas. Of women FH mutation carriers from 16 families, 100% (22/22) had uterine fibroids. Our study shows that expression of cutaneous manifestations in HLRCC ranges from absent to mild to severe cutaneous leiomyomas. FH mutations were associated with a spectrum of renal tumours. No genotype-phenotype correlations were identified. CONCLUSIONS: In combination with our previous report, we identify 31 different germline FH mutations in 56 families with HLRCC (20 missense, eight frameshifts, two nonsense, and one splice site). Our FH mutation detection rate is 93% (52/56) in families suspected of HLRCC.

Host-clonal interactions in the generation of proviral gene deletion variants.

A nonproducer clone (clone A1) (from a retrovirus-infected guinea pig fibrosarcoma) has been described that under conditions of in vivo immunologic selection forms variants that lack the proviral gene. One trivial explanation for the apparent loss of the provirus from clone A1 is that clone A1 did not originate from a single cell. For evaluation of this possibility, subclones were derived from clone A1 and tested for tumor recurrence in nonimmune and virus-immune animals. Each of four subclones contained the A1 provirus and exhibited specific viral interference; tumor recurrences formed from each of these four subclones lacked the clone A1 provirus. Possible, when heterogeneous populations of retrovirus-infected cells are injected into nonimmune animals, some clones will elicit immunologic responses to retroviral antigens and subject other clones in the population to immunologic selective pressures. For testing this concept, clone A1 was injected in admixture with a producer clone (clone A4) into nonimmune Sewall Wright strain 2 guinea pigs. Tumors formed in nonimmune guinea pigs inoculated with clone A1 in admixture with clone A4 were shown to lack a detectable clone A1 provirus. The results supported the concept that a somatic mutational event (deletion of the proviral gene) occurs during growth of clone A1. When heterogeneous populations of retrovirus-infected cells are injected into animals, host-clonal interactions may occur leading to outgrowth of proviral gene deletion variants. These results supported the notion that interactions between tumor clones and the host can change the dominant clonal type of the tumor and provide a genetic basis for this change.

Tumor suppression by pyran copolymer: correlation with production of cytotoxic macrophages.

Intraperitoneal administration of pyran copolymer (pyran-2-succinic anhydride-4,5-dicarboxytetrahydro-6-methylanhydride polymer) protected C3H/HeN male mice against tumor development after intradermal challenge with cells of the transplantable, methylcholanthrene-induced, syngeneic fibrosarcoma 1038. Peritoneal cells from pyran-treated but not from normal animals suppressed tumor development in local passive-transfer experiments. Adherent peritoneal cells from pyran-treated mice were cytotoxic in vitro to several syngeneic murine tumor cell lines including 1038.

Clonality of pulmonary metastases from the bladder 6 subline of the B16 melanoma studied by southern hybridization.

The possible clonal origin of pulmonary metastases from the bladder 6 subline of the B16 melanoma (B16-BL6) was evaluated with the use of the technique of Southern hybridization. Genetic markers were introduced into B16-BL6 cells by the transfection of a plasmid containing a bacterial gene. Genetic markers were detected by hybridizing DNA extracted from tumors with a 32P-labeled bacterial gene. Clones containing unique genetic markers were mixed and injected into the footpads of normal syngeneic C57BL/6 mice. Footpad tumors were amputated when they were 1 cm in diameter. Individual pulmonary metastases were harvested and expanded by implantation in nude mice. Although footpad tumors contained both genetic markers, virtually all (13/15) pulmonary metastases contained single unique genetic markers. Pulmonary metastases of both marker types were identified. Two examples of pulmonary metastases of mixed origin were detected. The Southern hybridization technique was sufficiently sensitive to detect a clone that was present at 5% of the total cell population.

Local antitumor activity of a primary and an anamnestic response to a syngeneic guinea pig hepatoma.

After intradermal (id) injection, the line-10 hepatoma grew progressively in nonimmune guinea pigs, whereas the line-1 hepatoma grew for approximately 2 weeks, developed central necrosis, ulcerated, and regressed. Growth of the line-10 hepatoma was suppressed when line-10 hepatoma cells were mixed with antigenically distinct line-1 hepatoma cells before id injection into syngeneic strain-2 guinea pigs. Mixture of line-10 with irradiated line-1 or viable strain-2 embryo cells did not inhibit tumor growth. Preimmunization of recipients to line-1 cells abrogated the suppression of tumor growth from mixtures of line-1 and line-10.

Failure of cryosurgical treatment of experimental intradermal tumors to eradicate microscopic lymph node metastases in guinea pigs.

Inbred Sewall Wright strain 2 male guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated either by local excision, cryosurgery, or intralesional injection of BCG. Cryosurgery and local excision were effective in eliminating growth of the primary tumor but failed to prevent growth of lymph node metastases. In contrast, intralesional injection of BCG caused regression of primary tumors and prevented growth of lymph node metastases.