Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials.

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.

The human T-lymphotropic virus type I tax gene can cooperate with the ras oncogene to induce neoplastic transformation of cells.

Epidemiologic studies have linked infection by the human T-lymphotropic virus type I (HTLV-I) with the development of adult T-cell leukemia. The low penetrance of the virus and the long latency for disease manifestation are factors that obscure the role of HTLV-I infection in oncogenesis. We have used an in vitro transformation assay system to determine directly whether the HTLV-I tax gene has transformation potential. Transfection of the tax gene alone into early-passage rat embryo fibroblasts did not induce morphological alterations. However, cotransfection of tax with the selectable marker plasmid pRSVneo gave rise to G418-resistant colonies that could be established as immortalized cell lines. Cotransfection of tax with the ras oncogene into rat embryo fibroblasts gave rise to foci of transformed cells that were highly tumorigenic in nude mice. These data represent a direct demonstration of the oncogenic potential of the tax gene in nonlymphoid cells and establish HTLV-I as a transforming virus.

The E1a gene of adenovirus type 2 reduces the metastatic potential of ras-transformed rat embryo cells.

We have previously demonstrated that second-passage rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells cotransformed with the ras oncogene and the adenovirus type 2 (Ad2) E1a gene are tumorigenic but either fail to metastasize or exhibit a very low metastatic potential. In this report, we demonstrate that transfection of the Ad2 E1a gene into four independent ras-transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to that of the parental cell line. Transfection of cDNAs for the 12S and 13S E1a transcripts showed that the 12S cDNA was highly effective in reducing the metastatic potential of ras-transformed cell lines, while the 13S cDNA showed an effect in only one of the two cell lines tested. This effect is specific to the Ad2 E1a gene, since ras-transformed cell lines expressing the Ad12 E1a gene maintained their high metastatic potential. We hypothesize that the Ad2 E1a gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.

Karyotypic analysis of diploid or near diploid metastatic Harvey ras transformed rat embryo fibroblasts.

The hypothesis of tumor progression proposed by Nowell [P. C. Nowell, Science (Wash. DC), 194: 23-28, 1976] states that one mechanism for the development of the metastatic phenotype could be the induction of chromosomal instability. We have developed a new experimental system for studying the induction of the metastatic phenotype using early passage fibroblasts which become metastatic in nude mice after transformation with the Harvey ras oncogene [R. J. Muschel et al., Am. J. Pathol., 121: 1-8, 1985; R. Pozzatti et al., Science (Wash. DC), 232: 223-227, 1986]. Since the early passage fibroblasts themselves are diploid, we reasoned that this might be a system in which karyotypic change after tumor formation or metastasis might easily be evaluated. Thus, we performed cytogenetic analysis on multiple metastases and tumors which had been derived from cells transformed with the cellular Harvey ras1 oncogene and compared their karyotypes. The karyotypes of the cells isolated from 5 tumors and 14 metastases were, as far as we could determine, identical to those of the injected cells. This could easily be evaluated because of the two clones studied one was diploid; the other has a trisomy of chromosome 4 without any other detectable abnormality. These results suggest that in this system using nude mice, selection for a necessary or even advantageous chromosomal aberration does not occur during tumor formation or metastasis. Furthermore, they indicate that the presence of the ras gene itself does not induce chromosomal rearrangements or aneuploidy and that a cell can be both tumorigenic and metastatic yet remain diploid.

Altered expression of NM23, a gene associated with low tumor metastatic potential, during adenovirus 2 Ela inhibition of experimental metastasis.

NM23, a novel gene associated with low tumor metastatic potential, has been investigated in an experimental system in which metastasis is inhibited by the transfection of viral and cellular oncogenes. The experimental system utilizes transfection of the Adenovirus 2 Ela gene to inhibit metastasis: rat embryo fibroblasts (REF) transfected with c-Ha-ras were highly metastatic, while REF cotransfected with ras and Ela were virtually nonmetastatic. NM23 RNA levels were higher in three independently ras + Ela-cotransfected, low metastatic REF lines than in three independently ras-transfected, highly metastatic REF line. Differences in hybridizable NM23 RNA levels between the two groups of transfected cell lines ranged from 2- to 8-fold. In situ hybridization demonstrated that the relatively high NM23 RNA levels in low metastatic ras + Ela-cotransfected REF cells were not due to overexpression of the NM23 gene by a subpopulation of cells. Thus, the metastasis-inhibitory effect of the exogenously added Ela gene has been associated with increased activation of the cellular NM23 gene. This associated is particularly significant in light of the very few changes observed in translatable steady-state RNA levels between ras- and ras + Ela-transfected REF lines. The data identify NM23 as a candidate for a gene that suppresses the malignant state.

Expression of matrix metalloproteinase genes in transformed rat cell lines of high and low metastatic potential.

The enzymes that comprise the family of matrix metalloproteinases (MMPs) share the capacity to degrade extracellular matrix components. A large body of evidence indicates that certain members of this metalloproteinase gene family play critical roles in determining the malignant phenotype of solid tumors. We previously have derived transformed cell lines with vastly different metastatic potentials by transfecting different combinations of oncogenes into primary rat embryo cells. Conditioned medium from those cell lines was assayed by Western blot analysis for the production of four separate matrix metalloproteinases to see whether a correlation could be found between protease expression and the metastatic phenotype. The transformed rat embryo cell lines with high metastatic potential were found to produce high levels of the stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) proteases, while the nonmetastatic lines produced low or undetectable levels of these two enzymes. No correlation was seen between the metastatic phenotype of the cell lines and the level of expression of two other matrix metalloproteinases, the M(r) 72,000 type IV collagenase (MMP-2) and the M(r) 92,000 gelatinase (MMP-9). These data suggest that the differential regulation of the stromelysin proteases may contribute to the difference seen in the metastatic potential of these cell lines.

Modulation of the transformed and neoplastic phenotype of rat fibroblasts by MHC-I gene expression.

Transfection of the activated ras oncogene (Ha-ras) into second passage rat embryo fibroblasts can induce the metastatic phenotype, while cotransfection of Ha-ras with the adenovirus type 2 E1a gene (Ad2-E1a) yields cells which are tumorigenic but nonmetastatic in nude mice. Because of the presence in nude mice of natural killer cells and B-lymphocytes, which might account for the different metastatic behavior of single versus double transfectants, we used triple deficient mutants as recipient animals in tumorigenicity assays. These mice carry two additional mutations resulting in the deficiency of natural killer cells and activated B-lymphocytes. We observed that the rat embryo fibroblast transfectants exhibit the same metastatic behavior in nude as well as in triple deficient mice, indicating that natural killer and B-cells are not responsible for the observed difference in metastatic phenotype between Ha-ras and Ha-ras plus Ad2-E1a transfectants. Double transfectants were found to express higher levels of major histocompatibility complex class I genes and the degree of expression appeared to correlate inversely with in vitro and in vivo parameters such as the ability to grow in agar-containing semisolid media and rate of tumor formation in triple deficient mice. Our observations are consistent with the concept that expression of major histocompatibility class I genes may be involved in regulating and modifying cell behavior by mechanisms independent of their role in immune recognition.

Secretion of type IV collagenolytic protease and metastatic phenotype: induction by transfection with c-Ha-ras but not c-Ha-ras plus Ad2-E1a.

Activated ras oncogene transfection into suitable recipient cells has been shown to induce the metastatic phenotype (Thorgeirsson, et al., Mol. Cell. Biol., 5: 259-262, 1985). We have used this model system to study the correlation of basement membrane collagenolysis with metastatic propensity. The c-Ha-ras oncogene alone, or combined with v-myc, transfected into early passage rat embryo fibroblasts, induce these cells to secrete high levels of type IV collagenolytic metalloproteinase and to concomitantly exhibit a high incidence of spontaneous metastases in nude mice. Cotransfection of c-Ha-ras plus the adenovirus type 2 E1a gene yields cells which are highly tumorigenic but nonmetastatic and fail to produce type IV collagenase. This effect is due to a suppression of collagenase elaboration, not increased production of a collagenase inhibitor, and not decreased production of a collagenase activator. The characteristics of the collagenase are identical to tumor type IV collagenase described previously. The nonmetastatic cells which failed to produce type IV collagenase retain the ability to secrete high levels of plasminogen activator. Transfection with the protooncogenic forms of Ha-ras or mos, or spontaneous transformation of NIH 3T3 cells or chemical transformation of BALB 3T3 cells yields cells which fail to produce collagenase, are tumorigenic, but totally nonmetastatic. These data support a biochemical linkage of type IV collagenase expression with the metastatic phenotype in this rodent system.

Allelic loss on chromosome 8p12-21 in microdissected prostatic intraepithelial neoplasia.

The development and progression of human prostate cancer is associated with genetic abnormalities in tumor cells. Inactivation of tumor suppressor genes due to allelic loss is thought to be an important mechanism of gene alteration in prostatic neoplasms. In this study we examined allelic loss on chromosome 8p12-21 in microdissected samples of normal prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and invasive prostate carcinoma from the same patients. Tissue microdissection under direct microscopic visualization procures pure populations of cells of interest, including small lesions such as PIN. Among 30 patients with concomitant cancer and PIN, we found loss of heterozygosity on chromosome 8p12-21 in 63% (34 of 54) of foci of PIN examined and 90.6% (29 of 32) of tumors, suggesting that abnormalities on chromosome 8p12-21 may be important in the early stages of prostatic carcinoma development. Several cases in which multiple foci of PIN from the same patient were sampled showed different patterns of allelic loss. Fifty-five % (16 of 29) of the prostate carcinomas contained a potential precursor PIN focus based on allelic loss pattern. Our results are consistent with the hypothesis that PIN arises multifocally within the prostate gland, and that a subset of these lesions progress to become carcinoma.

Analysis of 99 microdissected prostate carcinomas reveals a high frequency of allelic loss on chromosome 8p12-21.

To investigate the possible involvement of a tumor suppressor gene(s) on chromosome 8 in prostatic neoplasms, we performed a comprehensive loss of heterozygosity (LOH) study on 99 tumors from 97 prostate cancer patients. One of the carcinomas was a lymph node metastasis; the other 98 were primary carcinomas. Pure populations of carcinoma cells and normal epithelia were procured by tissue microdissection. Two separate tumor foci were obtained from each of two patients. Microsatellite markers from 25 loci on the short arm and one locus on the long arm of chromosome 8 were used for PCR-based LOH analysis on matched normal and tumor DNA samples. The overall LOH on 8p in this study was 85.9% (85 of 99) of carcinomas. The loss was highest at markers D8S133, D8S136, NEFL, and D8S137 (62,72, 64, and 75%, respectively), which are located at 8p12-21. Seventy-nine of 99 tumors exhibited loss in at least one of these four loci. In contrast, LOH at 8p22 was much lower: 17,18,18, and 19% at D8S549, D8S602, D8S254, and D8S261, respectively, with 25 of 99 tumors showing deletion in one or more of the four loci. All but 5 tumors with deletions in this more distal region had at least one retained locus between the 8p22 deletion and a more proximal region of loss at 8p12-21; 1 tumor had loss at 8p22 but not 8p12-21. This suggests there may be two distinct regions of loss and, therefore, two tumor suppressor genes on this chromosomal arm. The loss on 8p12-21 showed little or no correlation with grade or stage of disease.

Progelatinase A mRNA expression in cell lines derived from tumors in patients with metastatic renal cell carcinoma correlates inversely with survival.

OBJECTIVES: Tumors are thought to metastasize by a process involving tumor cell attachment to extracellular matrix, degradation of matrix components by tumor-associated proteases, and cellular movement into the area modified by protease activity. Type IV collagen comprises the major element tumor cells must degrade to gain access to the rest of the body. Renal cancer cell line progelatinase A (E.C. 3.4.24.24; 72-kDa type IV collagenase; MMP-2) mRNA expression was correlated with patient survival. METHODS: Total cellular mRNA was extracted from tumor cell lines derived from patients with metastatic renal cell carcinoma. The results of the densitometric analysis of Northern blots were correlated with patient survival. Formalin-fixed, paraffin-embedded tissue sections of primary renal cancers were examined for immunohistochemical expression of MMP-2. RESULTS: Cell lines established from 23 primary renal tumors and six metastatic sites in 26 patients with metastatic renal carcinoma were studied. Variable expression of progelatinase A, relative to A2058 melanoma cells (mean +/- SEM, 0.60 +/- 0.21; median, 0.082; range, 0 to 4.78), was found. There was a significant inverse association between patient survival and the log of the MMP-2 expression (P = 0.045 by the Cox proportional-hazards model). Using a cutoff value of 0.10, the closest round number to the median expression of MMP-2, a significant difference between survival of patients with lower and higher MMP-2 expression in their primary renal cell line was found (P = 0.0054). Cell lines with low, intermediate, and high expression of MMP-2 mRNA all had primary tumors with high tissue immunohistochemical expression of MMP-2. CONCLUSIONS: These studies demonstrate an inverse relationship between renal cancer cell line MMP-2 mRNA expression and patient survival. Immunohistochemical studies of the primary tumors from which the cell lines were derived uniformly showed high MMP-2 expression. Previous work suggests local renal factors upregulate cellular expression of MMP-2 in the primary tumor, and are not active at extrarenal sites.

Regulation of the metastatic phenotype by the E1A gene of adenovirus-2.

We have previously demonstrated that rat embryo cells transformed by the ras oncogene alone are both tumorigenic and highly metastatic when injected into nude mice. In contrast, rat embryo cells transformed with the ras oncogene and the adenovirus 2 (Ad2) Ela gene are tumorigenic but either fail to metastasize, or exhibit a very low metastatic potential. Here we demonstrate that transfection of the Ad2 Ela gene into several of the ras transformed rat embryo cell lines results in a dramatic reduction in metastatic potential relative to the parental cell line. Transfection of cDNAs for the 12S and 13S Ela transcripts showed that both gene products are capable of reducing the metastatic potential of the ras transformed cell lines, however the 12S cDNA was more effective. This effect is specific to the Ad2 Ela gene as ras transformed cell lines expressing the Ad12 Ela gene or the human N-myc gene maintained their high metastatic potential. We hypothesize that the Ad2 Ela gene may regulate the expression of one or more cellular genes that contribute to the metastatic phenotype.

Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus.

A series of recombinant molecules were constructed which direct the expression of the easily assayed gene chloramphenicol acetyltransferase. We have used these recombinants to show that the 73/72-base-pair tandem repeat unit from the Moloney murine sarcoma virus long terminal repeat shares a number of properties with the prototypic enhancer element, the simian virus 40 72-base-pair repeat. Specifically, the Moloney murine sarcoma virus sequence significantly enhances the level of gene expression at both 5' and 3' locations and in either orientation relative to the test gene. It is able to enhance gene activity both from its own promoter and from a heterologous (simian virus 40) promoter. The 73/72-base-pair subunits of the Moloney murine sarcoma virus enhancer differ in sequence by four nucleotides and also in the strength of their enhancer function. The promoter distal A repeat is at least three times as active as the promoter proximal B repeat in enhancing chloramphenicol acetyltransferase expression. Results of these studies also show that the enhancer sequence alone is unable to induce gene activity but requires other promoter elements, including a proximal GC-rich sequence and the Goldberg-Hogness box.

The location of the polio genome protein in viral RNAs and its implication for RNA synthesis.

Evidence is presented that a small protein (VPg) is covalently attached to the 5'-terminal oligonucleotide VPg-pU-U-A-A-A-A-C-A-Gp of the polio genome, to nascent strands of the polio replicative intermediate and to poly(U) of minus strands. A model of polio RNA replication is proposed implicating VPg in initiation of RNA symthesis, possibly as primer.

Purine mutants of mammalian cell lines: III. Control of purine biosynthesis in adenine phosphoribosyl transferase mutants of CHO cells.

Spontaneous and mutagen-induced 2,6-diaminopurine-resistant mutants of Chinese hamster ovary (CHO-K1) cells were isolated. Such mutants fell into two classes: spontaneous and ethylmethane-sulfonate-induced mutants had approximately 5% wild-type adenine phosphoribosyl transferase (APRT) activity, whereas ICR-170G-induced mutants had barely detectable APRT activity. Since it has been reported that human hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (Lesch-Nyhan syndrome) and APRT mutants over-produce purines, we examined the control and rate of purine biosynthesis in the Chinese hamster mutants. End product inhibition by adenine could not be demonstrated in such mutants, indicating that the active feedback inhibitor is a nucleotide rather than the free purine base, HGPRT activity was normal in all mutants examined except in one isolate. Purine biosynthesis as measured by the accumulation of the purine biosynthetic intermediate phosphoribosyl formylglycineamide was not elevated in the mutants as might have been predicted from work with Lesch-Nyhan cells. The data also suggest that our strain of CHO-K1 is physically or functionally haploid for the APRT locus.

Expression of nm23 in cell lines derived from patients with metastatic renal cell carcinoma.

Reduced expression of nm23 has been associated with increased metastases and decreased survival in a variety of malignancies. In the present study, the expression of nm23 was examined by Northern and Western blot analyses in a series of cell lines derived from patients with metastatic renal cell carcinoma. Two of twelve (17%) informative cell lines derived from 9 patients had loss of heterozygosity at Nm23-H1. Twenty-two renal cancer cell lines derived from primary tumors, 5 cell lines derived from metastatic tumors and 4 short-term cultures of normal proximal renal tubular cells all expressed Nm23 mRNA in varying amounts. On average, the level of expression of Nm23 mRNA in short-term cultures of benign proximal renal tubular cells was found to be similar to the level seen in renal cancer cell lines. Twenty-eight cell lines derived from renal primary tumors and 8 cell lines derived from metastatic tumors expressed both the Nm23-H1 and Nm23-H2 proteins. High or low relative expression of nm23 at the mRNA or protein level did not correlate with survival. The absence of any anomalous pattern of expression of the nm23 genes and the lack of correlation of expression with survival suggests that nm23 does not play a central role in the progression of this tumor type.

Chromosome 16 allelic loss analysis of a large set of microdissected prostate carcinomas.

PURPOSE: To perform loss of heterozygosity (LOH) analysis on chromosome 16 in 102 highly purified DNA samples isolated from one or more adenocarcinomas, prostatic intraepithelial neoplasia (PIN), and matched benign prostatic epithelium from 95 radical prostatectomy patients. MATERIALS AND METHODS: Specimens were procured by microdissection of frozen tissue samples, thus ensuring that highly select pure populations of cells were obtained for DNA extraction and LOH analysis. Multiple microsatellite markers were used to determine allelic loss on chromosome 16q. RESULTS: Overall loss on 16q was seen in 31% of the cancers, and occurred more frequently in high stage cancers than low stage cancers. In contrast, allelic loss in PIN failed to exceed 6% at any of the loci that were examined. CONCLUSIONS: These results suggest that inactivation of a putative tumor suppressor gene on 16q may be involved in the progression of some prostate cancers.