High expression of shMDG1 gene is associated with low metastatic potential of tumor cells.

Metastasis is the primary cause of mortality associated with cancer. Molecular mechanisms leading to metastatic spread are poorly studied. To get a better understanding of this process, we compared the gene expression pattern of two isogenic cell lines, HET-SR and HET-SR1 (Rous Sarcoma Virus-transformed embryo hamster fibroblasts) with different metastatic activity using the differential display technique. A novel cDNA of hamster gene shMDG1 (Syrian hamster homologue of microvascular differentiation gene 1), which had 94% homology with rat MDG1 gene, was identified. Expression of shMDG1 was increased in low metastatic HET-SR cell line in comparison to high metastatic HET-SR1. Sequence analysis of the ORF of shMDG1 gene showed that it belongs to the DnaJ/heat-shock proteins of 40 kDa (HSP40) chaperones family, considered to function as a cochaperone of HSP70 family. In order to confirm involvement of shMDG1 in metastasis, we injected parental and shMDG1 overexpressed cells into animals. We showed that overexpression of the shMDG1 gene significantly diminished the metastatic activity of both HET-SR and HET-SR1 cells. The shMDG1-induced repression of metastasis was not connected with alterations in cell proliferation and motility in vitro, but correlated well with a decrease in content of the Asn-linked beta1-6 branched oligosaccharides on cell surface.

C-terminal end of v-src protein interacts with peptide coded by gadd7/adapt15-like RNA in two-hybrid system.

The significant differences in the metastatic properties of hamster fibroblasts transformed by the Rous sarcoma virus (RSV) were associated with mutations in the v-src carboxy-terminal region. To identify the capacity of this region for protein-protein interaction the two-hybrid system was used. The cDNA clone (vseap1), producing the protein specifically bound with the v-src C-terminal part in yeast cells in vivo and in GST-fusion system in vitro was isolated. Vseap1 shared 68% of homology with stressful agents induced RNA-gadd7/adapt15. Two vseap1 specific messenger RNAs were identified: 0.9-kbp RNA expressed in all transformed cells and three times less in embryo fibroblasts; 3.1-kbp transcript was deleted in the cells with suppressed v-src activity and H2O2 resistance.

Phosphorylation of p125FAK and paxillin focal adhesion proteins in src-transformed cells with different metastatic capacity.

Hamster fibroblasts transformed by Rous sarcoma virus (RSV) display different metastatic potentials that are associated with specific structural features of the v-src oncoprotein. This diverse metastatic activity could be due to various tyrosine phosphorylation levels of specific src protein substrates. To check this hypothesis, phosphorylation of the FAK and paxillin proteins, involved in signal transduction pathways and known as src protein substrates, was tested. It was shown that FAK and paxillin are hyperphosphorylated in the high metastatic cell lines as compared with the phosphotyrosine level of these proteins found in the low metastatic cell lines. In addition, our data confirm that v-src protein plays a direct role in paxillin phosphorylation.

Simultaneous detection of genetic and immunological markers in non-small cell lung cancer: prediction of metastatic potential of tumor.

The restriction fragment length polymorphism of c-Ha-ras-1 and L-myc genes and expression of cell surface effector molecules were studied to determine their potential utility as markers for assessing risk of metastasis in 84 lung cancer patients. We performed a comparative study of primary lung carcinomas, metastases, adjacent tissues and blood samples in a group of patients with lung cancer of different histological types, grade of differentiation and presence of regional and distant metastasis. No differences in the frequency of c-Ha-ras-1 rare alleles were found between lung cancer patients and unaffected controls. The detection of common a4-allele seems to be associated with metastasis and low differentiation of lung carcinomas. S-allele of L-myc was observed in 82.6% of patients with metastatic lesions. Homozygosity of L-allele patients was not evidence for distant metastasis and only 17.4% of these patients have metastatic lesions of the lymph nodes. The expression of HLA class I and receptor of transferrin (TrRec) were tested immunohistochemically in the same patients. In the group of squamous cell carcinomas with regional metastases the expression of HLA class I antigens was decreased [7/21 (33.3%) positive staining tumors versus 13/20 (65.0%) in the group without metastases]. The opposite situation was observed for TrRec. The data of restriction fragment length polymorphism of oncogenes and expression of two cell surface effector molecules, identified in the same patients, were combined. The registration of more than one poor marker, tested in individuals with squamous cell carcinoma, closely correlated with dissemination and advanced stage of the disease. Nearly 90% (20/22) of patients with well and moderately differentiated tumor revealed metastatic lesions versus 6.6% (1/15) of patients with manifestation of a single poor marker. Finally, proposals could be made for the development of a risk group that incorporates both clinical and molecular biology features in the prediction of metastasis.

C-ets-1 protooncogene expression alters the growth properties of immortalized rat fibroblasts.

Ets family genes have been cloned and characterized from a variety of species ranging from human to Drosophila. The ets proteins encode transcription factors that activate transcription via specific binding to GGAA core sequence present in various promoter/enhancers. To investigate the role of ets protooncogene expression on the growth properties of rat embryo fibroblasts (REF), we constructed and introduced ets expression vectors into primary, as well as immortalized REF cells. The transfected cells contained multiple copies of the vector DNA, and the Northern blot analysis demonstrated overexpression of the c-ets-1-specific mRNA. Although the expression of the ets genes was unable to immortalize primary rat embryo fibroblasts, the expression of ets-1 in REF-1 cells enabled their growth in serum-free medium and effected tumorigenic activity in nude mice.

Differences in the organization and methylation patterns of integrated avian sarcoma proviral DNA sequences in nonpermissive and permissive mammalian cells.

Four types of avian sarcoma virus (ASV)-transformed mammalian cells were analyzed for the presence of ASV-specific sequences in their genome DNA. A great variability in the number of proviral copies and their structure within the DNA of these lines was observed. In all cells tested gag and src sequences were present in a flexible arrangement. The greatest variation was detected within proviral sequences corresponding to pol and env regions. The number of integrated ASV proviral copies do not correlate with the capability of these cells to produce viral particles. Virus-producing (K2S and K12) and virogenic (XC) cells contain in their chromosomal DNA at least one complete proviral genome, whereas proviral sequences in helper-dependent nonvirogenic cells are substantially changed. Provirus expression level does not correlate with the number of integrated virus copies. In the non-virus-producing cells the proviral sequences are hypermethylated.

BrdU-induced changes in the provirus of PR-RSV in mammalian cells.

Previously we demonstrated differences in the organization and methylation pattern of integrated Rous sarcoma proviral sequences in helper-dependent virogenic rat TWERC cells. In the present study we attempted to induce changes in the integrated viral genome of TWERC cells using 5-bromodeoxyuridine (BrdU). Four clones (A, B, C, and F) derived from the parental cell line were treated for 10 months with different concentrations of BrdU. Restriction enzyme analysis of the parental cell line and its clones showed that the cells contained in their genomic DNA two copies of deleted provirus. In TWERC cells, the PR-RSV provirus lost the whole env gene and part of the 3' end of the pol gene. The proviral sequences in DNA from the TWERC-derived clones were found to be hypermethylated. A slightly different situation was seen in clone C where demethylation of the provirus in the 3' region and in the 5' end of the genome was found. The level of mRNA expression both in the parental cells and in the clones correlated with the methylation pattern of the PR-RSV provirus. Clone C was less methylated and expressed more virus-specific RNA. The possible role of BrdU in these events is discussed.

Differential expression of cellular genes in Rauscher leukaemia.

The methods of hybridization in solution and blot hybridization showed that spleen cells from BALB/c mice contain "silent" genes which can amplify and change their structure after infection by Rauscher leukaemia virus. The "silent" gene product is nuclear 35S RNA detectable by comparative electrophoretic analysis of the heterogeneous nuclear RNA from leukaemic and normal cells. About 7% of this 35S RNA is represented by the virus-specific sequences, but a major part is represented by the cellular sequences. In order to study the expression of the sequences homologous to 35S RNA in leukaemic and normal cells, hybridization in solution was used. Expression of the complete copies of 35S RNA was observed in nuclei of virus-infected cells, whereas this RNA in the cytoplasm is represented by the incomplete copies. The expression of the sequences homologous to this 35S RNA in normal mouse spleen cells was not revealed.

Kinases of the Src family: structure and functions.

Tyrosine kinases of the Src family are involved in different signal transduction pathways in cells. The corresponding genes participate in such vital processes as growth, differentiation, adhesion, transcription, etc. Specific structural changes confer oncogenic properties to the Src protein. In this review, we summarize the available data on the structure, substrates, regulation mechanisms, and role of nonreceptor tyrosine kinases by the example of the src gene product (as the prototype member of this family) and a number of related proteins.

RSV-transformed mammalian cells: an analysis of RNAs specific for exogenous and endogenous viruses.

A marked decrease in expression of RSV-specific sequences was observed in cryptovirogenic RSV-transformed RVP3 murine cells during passaging. Simultaneously an increase in expression of sequences specific for C-type murine viruses was observed both in these cells and other RSV-transformed murine cells. No activation of C-type viral genome was detected in spontaneous murine lung tumors and in methylcholanthrene-induced murine tumors. Four types of virus specific RNAs were found in XC cells: the 30--35 S, the 26--28 S, the 21--23 S and the 15--18 S type. The 9--14 S RNA was the dominant species of RSV-specific RNA in TWERC cells.

Two consistently deleted regions within chromosome 1p32-pter in human non-small cell lung cancer.

Allelic losses at 1p32-pter have been reported as frequent events in human non-small cell lung cancer (NSCLC). To further characterize the region of deletions, we studied loss of heterozygosity on a panel of 102 microdissected NSCLC samples with 20 polymorphic markers spanning 1p32-pter. Two shortest regions of the overlap of the deletions (SROs) were found: SRO 2a (D1S417--D1S57) and SRO 2b (D1S450--D1S243). Allelic losses at either region correlated independently with advanced stage of disease and with postoperative metastasis and relapse (P < 0.05), suggesting that crucial genes in these regions are involved in NSCLC progression. Mol. Carcinog. 30:151--158, 2001.

Detection of virus-specific sequences in Drosophila melanogaster mutants induced by injection of RSV DNA into early embryos.

The injection of purified Rous sarcoma virus (RSV) (Prague strain) into Drosophila melanogaster (Oregon R line) eggs changes the fly phenotype in certain cases, and RSV-specific sequences can be identified in the Drosophila genome (ref. 1 and preceding paper). Here we have used Southern blotting to analyse in greater detail the proviral DNA present in several mutant lines of D. melanogaster produced by microinjection of intact RSV or plasmid DNA containing the viral insert. In certain populations of flies, RSV provirus was found to be incorporated into cellular DNA, and in one mutant family the unintegrated form of plasmid DNA was identified. Generally, the presence of injected genetic material in fly cells correlated with morphological changes in Drosophila.

Genetic effects of injection of Rous sarcoma virus DNA into polar plasm of early Drosophila melanogaster embryos.

Retroviral proviruses and the transposable elements of eukaryotic genomes are structurally similar. The biological significance of eukaryotic transposable elements has not been examined extensively but it is known that, like prokaryotic transposons, these elements can induce mutations in adjacent genes and cause their transposition. It is of interest to determine whether retroviral proviruses have the same mutagenic and gene transposing ability as transposable elements, particularly because the retrovirus genome is assumed to have originated from transposable elements of lower eukaryotes. The transfer of DNA sequences into animal zygotes or embryos by microinjection is a promising experimental approach for eluxidating their functions: when foreign DNAs were introduced into a mouse germ line, mutations were induced and at least in some mice, the mutation was caused by the insertion of a retroviral sequence. We have introduced Rous sarcoma virus (RSV) DNA into a germ line of Drosophila melanogaster, and describe here the resultant genetic effects.

Somatic genetic alterations (LOH) in benign, borderline and invasive ovarian tumours: intratumoral molecular heterogeneity.

Loss of heterozygosity (LOH) affects a number of chromosome regions in ovarian cancer, pointing to the possible involvement of tumour-suppressor genes in ovarian tumorigenesis. We performed comparative analysis of allelic loss at 6 frequently affected chromosome regions in a panel of 53 benign, borderline and malignant ovarian tumours. Precursor lesions could provide evidence that an accumulation of genetic events is required for normal ovarian epithelium to generate malignant tumours. LOH on chromosome 1p was relatively common in benign, borderline and malignant tumours, while at 11p and 7q it was observed not only in invasive but also in borderline tumours. Moreover, 17q and 18q were affected mainly in advanced malignant tumours and revealed a high frequency of clonal intratumoral heterogeneity. We encountered different spectra of genetic alterations in primary tumours and their metastasis, which may be the results of intratumoral heterogeneity leading to dissemination in only some sub-clones.

Allelic imbalance and instability of microsatellite loci on chromosome 1p in human non-small-cell lung cancer.

The mapping of allelic loss on the short arm of chromosome 1 has been performed in non-small-cell lung cancer. We used a set of 11 microsatellite loci spanning 1p to examine the frequency of allelic imbalance in a panel of 58 tumours. Fifty-one of 58 (87.9%) cases have shown somatic allelic loss at one or more loci tested. The two shortest regions of the overlap (SRO) of the deletions have been identified: SRO 1 at 1p13.1 and SRO 2 at 1p32-pter. Allelic losses at these regions have been compared among adenocarcinoma and squamous cell carcinoma and no difference has been found. In contrast to SRO 1, deletions at SRO 2 significantly correlated with advanced stage of the disease as well as post-operative metastasizing and relapse. These data may suggest that SRO 1 and SRO 2 can harbour tumour-supressor genes (TSGs) involved in different stages of NSCLC development. SRO 2 is still quite large and its refined mapping should help attempts to clone and identify the putative TSG(s). Microsatellite instability (replication errors) affecting only 6 (10.3%) of 58 tumour samples is an infrequent genetic alteration at the loci tested.

Unstable visible mutations induced in Drosophila melanogaster by injections of oncogenic virus DNA into the polar plasm of early embryos.

When RSV DNA cloned in pBR 322 or DNA of simian adenovirus Sa7 (C8) is injected into the pole plasm of embryos of various Drosophila stocks, the progeny of 1-70% of the surviving flies display visible mutations. The mutagenesis is partially directed: the loci mutating due to retrovirus and adenovirus DNA do not overlap. The majority of resulting mutants are characterised by high instability: reversions and new mutations occur in them, which sometimes spread over the whole population ("explosive" instability). The injected sequences are revealed by dot-hybridization in the DNA of many mutant strains, but only rarely by Southern blotting procedures. The results show that the microinjection of oncovirus DNA into embryos is an approach for obtaining highly unstable strains even from wild-type stable Drosophila stocks without crosses with MR lines or the introduction of P elements. The sets of unstable mutations induced by oncovirus DNA is different from those in hybrid dysgenesis.

Clustering of discrete cell properties essential for tumorigenicity and metastasis. III. Dissociation of the properties in N-ras-transfected RSV-SR-transformed cells.

We have previously shown that RSV-SR-transformed hamster cells acquire high resistance to H2O2, i.e. the cytotoxic product of activated macrophages (H2O2R) and that they begin to secrete PGE (PGES), thus inactivating the CTA of NK cells. Among normal cells, the same phenotype is expressed in activated macrophages. In all our RSV-transformed cells these 2 properties were jointly expressed and correlated with high tumorigenicity and experimental metastasizing of these cells. We now show that transfection of 3 RSV-SR-transformed cell strains with activated N-ras leads either to complete inhibition of the H2O2R + PGES phenotype in all clones of one strain, or to inhibition of PGES only in the majority of clones of 2 other strains. Unexpectedly, the complete or partial inhibition of this phenotype did not alter the high tumorigenicity of 2 strains of these cells, but lower tumorigenicity was evident in almost all clones of the third strain (as well as in some gene-neo-transfected clones of these strains). The loss of PGES made these cells susceptible to the CTA of NK cells, while the loss of H2O2R did not alter their resistance to the CTA of macrophages. Expression of the H2O2R + PGES phenotype was retained in all cloned variants of control, gene-neo-transfected cells. The possible relation of the N-ras gene to regulation of src gene activities in RSV-SR-transformed cells is discussed.

A novel group IIA phospholipase A2 interacts with v-Src oncoprotein from RSV-transformed hamster cells.

We have isolated a novel isoform of phospholipase A(2). This enzyme was designated srPLA(2) because it was discovered while analyzing the proteins interacting with different forms of the v-Src oncoproteins isolated from Rous sarcoma virus-transformed hamster cells. It contains all the functional regions of the PLA(2) group IIA proteins but differs at its C-terminal end where there is an additional stretch of 8 amino acids. The SrPLA(2) isoform was detected as a 17-kDa precursor in cells and as a mature 14-kDa form secreted in culture medium. A direct interaction of the 17-kDa precursor with the Src protein was observed in lysates of transformed cells. Both the 17- and 14-kDa forms were found to be phosphorylated on tyrosine. To our knowledge, this is the first report of a PLA(2) group II protein that is tyrosine phosphorylated. We surmise that srPLA(2) interacts with the Src protein at the cell membrane during the process of its maturation.

Two novel variants of the v-src oncogene isolated from low and high metastatic RSV-transformed hamster cells.

Four different transformed cell lines were isolated as a result of independent infection of primary hamster fibroblasts by Rous sarcoma virus (RSV SR-D stocks). These lines differ by the level of their spontaneous metastatic activity: HET-SR-1, HET-SR-8, and HET-SR-10 cell lines induced 70-200 metastatic nodules in the lung and/or lymph nodes of inoculated animals (high metastatic lines, HM). Metastatic activity was not identified after injection of HET-SR cells (low metastatic line, LM). All cell lines contained one copy of integrated and expressed intact RSV provirus. The difference in the amount of v-src protein in cell lines was not correlated with their metastatic potential in vivo. Complete v-srcHM and v-srcLM genes were cloned from corresponding gene libraries and sequenced. In the unique region of both v-src isoforms a GC-rich insert of 60 nucleotides (20 a.a.) was found. The presence of this insert explains the unusual apparent molecular weight of protein encoded by v-srcHM and vsrcLM: 62 kDA. Both genes had 10 identical amino acid changes when compared to the known RSV SR-D v-src sequence. v-srcHM and v-srcLM differ by several amino acid changes. Most of them are localized in the unique domain and the extreme carboxy-terminal region of the of the oncoprotein. Both v-src variants and chimeric v-src with mutually substituted parts were subcloned in a retroviral vector and introduced into avian neuroretina cells. Significant differences in the morphology of transformed neuroretinal cells were associated with the mutations in the carboxy-terminal region of the v-src oncogene. Low metastatic HET-SR cells transfected with v-srcHM and the chimeric gene v-src-LH remarkably increased their metastatic potential. In contrast, this effect was not observed when the same cells were transfected with v-srcLM and the chimeric v-srcHL gene. Specific changes in the distribution of fibronectin matrix typical for high metastatic cells were found in the lines transfected with v-srcHM.

Oncogene expression in human tumors.

The expression of 9 oncogenes in primary tumors and in human tumors passaged in nude mice was tested: a total of 28 tumor types was analyzed. Oncogenes src, fps, and mos were not expressed in any of the tumors tested but oncogene myc was transcribed in most of the tumors and myc was over-expressed in 3 tumors passaged in nude mice (Ewing sarcoma, large intestine carcinoma and kidney carcinoma) and in primary fibrous histiocytoma. Enhanced transcription of ras and fos genes was observed nonspecifically in different tumors. Oncogene sis was activated specifically in metastases of different tumors in lymph nodes.