Relationship between intrinsic radiation sensitivity and metastatic potential

PURPOSE: Prior studies emphasized genetic modulation of tumorigenicity, and experimental metastatic potential in cells transfected with oncogenes. Whether the intrinsic radiaton sensitivity of cells might correlate with parallel changes in metastatic potential is unknown. METHODS AND MATERIALS: Rat embryo cells (REC) were transfected with the following oncogenes, and where appropriate, with corresponding selection markers: pCMVneopEJ6.6ras, pEJ6.6ras/v-myc, pEla, and pEJ6.6ras/Ela. Individual transfectant clones and corresponding pooled cellular populations were propagated in selective medium. In vitro cellular radiation sensitivity was determined via clonogenic assays, a minimum of three, by standard techniques and individual SF2 and MID parameters determined. Tumorigenicity was defined as the number of tumors forming following the injection of 1 x 10(5) - 1 x 10(6) cells into the axillary pouch of three different strains of immune-deficient mice. Animals were killed once resultant tumors reached a maximum size of 1.5-2.0 cm in maximum diameter. For determination of experimental metastatic potential, between 1 x 10(5) - 1 x 10(6) cells were injected into the tail veins of litter-matched sibling mice in parallel to the tumorigenicity studies. RESULTS: Radiobiologic studies indicate similar levels of radiation sensitivity among REC, mock-transfected REC, Ela, and combined E1a/ras transfectants. pEJ6.6ras, and combined ras/myc transfected pooled cellular populations demonstrated increases in radiation resistance when compared to the pooled radiobiologic data from untransfected and mock-transfected corresponding pooled cellular populations (p <0.05, two-tailed test, SF2, MID). Rat embryo cells, Ela, and mock-transfectants were relatively radiation sensitive and nontumorigenic. pEla/ras was tumorigenic but demonstrated relatively low experimental metastatic potential. Ras, and ras/myc transfectants, demonstrated similar levels of experimental metastatic potential on lung colonization assays. CONCLUSIONS: A good correlation exists between the intrinsic radiation sensitivity and the experimental metastatic potential of transfected REC. The highest levels of radiation resistance in vitro and experimental metastatic potential in vivo were found among REC transfected with ras/myc or activated ras alone. E1a/ ras cotransfected cellular populations, although tumorigenic, were relatively radiation sensitive and nonmetastatic. Further study is needed to formulate a mechanistic explanation for the intriguing correlation between intrinsic radiation sensitivity in vitro and metastatic potential in vivo.     

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.     

Anti-tumor surveillance of non-contact-inhibited transformed cell lines

In order to determine if the correlated expression of transformation and tumorigenicity is affected by the agents used to induce transformants or by the immune status of the host used to test the tumorigenicity of transformants, we derived a series of cloned cell lines from foci of transformed cells induced by treatment of the contact-inhibited mouse cell line B/C-N7.ICI with the DNA demethylating agent 5-azacytidine (5-AZC) or the DNA demethylating and mutating agent benzo(a)pyrene dihydrodiol epoxide (BPDE). The transformed cell lines were injected into syngeneic nude and normal mice to determine their tumorigenicity. The results of this analysis showed that 93% of the transformants induced by 5-AZC treatment grew as tumors when injected into nude mice. Of those lines capable of growing as tumors in nude mice, 86% were also tumorigenic when injected into normal mice. In contrast, only 64% of BPDE-induced transformants grew as tumors in nude mice, and of those, only 44% were also tumorigenic in normal mice. The existence of non-contact-inhibited transformants that are tumorigenic only in nude mice indicates that host anti-tumor immune surveillance mechanisms are operative in normal mice. Further, the difference in both the percentage of transformed cell lines that are tumorigenic and the percentage of tumorigenic transformants that are susceptible to immune surveillance when transformants are induced by BPDE as compared to 5-AZC indicates that the transforming agent can affect both the correlation between the expression of transformation and tumorigenicity, and the interaction between the immune system and tumorigenic transformants.     

Human herpesvirus-6: tumorigenicity and tumor infiltrating lymphocytes.

We previously reported that human herpes-virus-6 (HHV-6) genome (strain GS) and a cloned subfragment (pZVH14) transfected NIH 3T3 cells, induced foci of transformation with a frequency significantly above the background level. The transformed cells produced tumors in nude mice and immunocompetent (Swiss) mice. In the current study, nude mice tumors were passed into Swiss mice and more aggressive tumors (G-2TS and 14-2TS derived from HHV-6 genome and pZVH14 DNA, respectively) were produced. 14-2TS tumors caused lung metastasis upon intravenous injection. In the case of subcutaneously growing aggressive tumors, tumor infiltrating lymphocytes (TIL) were isolated and characterized as T cells but lacked tumor specific killing as monitored by 51Cr-release assays. TIL lost activity at day 52 in a 4 h assay (but not in a 19 h assay) against the autologous tumor, but not a Maloney virus induced tumor (Yac-1). These studies indicate that an established nontumorigenic fibroblast cell line, when transfected with pZVH14 DNA of HHV-6, acquires both tumorigenic and metastatic potential and tumor bearing hosts mount immune response against such tumors.     

Transformation of human mammary epithelial cells by oncogenic retroviruses

We have introduced viral oncogenes into human mammary epithelial cells through the use of murine retroviruses. A continuous cell line (184A1N4) derived from benzo(a)pyrene treatment of normal breast epithelial cells was used as a recipient for the ras, mos, and T-antigen oncogenes. Each of these oncogenes enabled the 184A1N4 cells to grow in a selective medium, thus demonstrating the potential utility of these cells for oncogene detection and isolation. 184A1N4 cells transformed by T-antigen were nontumorigenic in athymic mice, but v-ras transformants were weakly tumorigenic. Transformants bearing both the T-antigen and ras oncogenes were strongly tumorigenic, however. The karyotype of these double transformants shows a high degree of stability. These results demonstrate the stepwise acquisition of the fully malignant phenotype by normal human epithelial cells in vitro.     

Lovastatin, a cholesterol biosynthesis inhibitor, inhibits the growth of human H-ras oncogene transformed cells in nude mice

Post-translational modification of oncogenic p21ras proteins with farnesyl, a lipid intermediate in cholesterol biosynthesis, is required for p21ras membrane association and for the ability of p21ras to transform cultured cells. We have tested the ability of lovastatin, a specific inhibitor of cholesterol biosynthesis, to inhibit the growth of ras oncogene-transformed cells in vivo. Balb/c mouse 3T3 cells, transfected with H-ras oncogene from human EJ bladder carcinoma, were highly tumorigenic in nude mice. Immunoprecipitation studies with transformed EJ cells showed that lovastatin (1-100 microM) inhibited p21ras membrane association in a concentration-dependent manner and that a 10 microM concentration reduced the amount of p21ras bound to the membrane by 50%. Lovastatin also inhibited EJ cell growth in a concentration range that closely paralleled that required for inhibition of p21ras membrane association. Treatment of nude mice bearing subcutaneous (s.c.) EJ tumors with lovastatin (50 mg/kg) significantly inhibited the abilities of these tumors to grow as early as four days and, by day 12, the lovastatin treated group of animals had tumors with an average size that was 3-fold smaller than those in the saline treated group. Western blotting studies showed that lovastatin (50 mg/kg) was also able to inhibit p21ras membrane association in EJ tumors implanted s.c. in nude mice. These results demonstrate that lovastatin, an inhibitor of cholesterol biosynthesis, inhibited in vivo tumor growth of H-ras oncogene transformed cells. The results also suggest that inhibition of p21ras membrane association, an essential step in ras oncogene neoplastic transformation, is one mechanism by which lovastatin may express its antitumor activity.     

Detection and identification of activated oncogenes in human skin cancers occurring on sun-exposed body sites

High-molecular-weight DNA isolated from eight fresh human skin cancers occurring on sun-exposed body sites were assayed for their ability to transform NIH 3T3 cells. A cotransfection protocol using pSV2-neo DNA, which confers resistance to the antibiotic G418, was used to select cells that had taken up the transfected DNA. About 2 weeks after transfection, G418-resistant colonies were pooled and injected s.c. into athymic nude mice. The NIH 3T3 cells transfected with DNA from six of the human skin cancers induced tumors in nude mice. DNAs from all six tumor cell lines contained human alu sequences. Southern blot hybridization with ras-specific probes revealed that DNAs from the four alu-rich tumors contained the human Ha-ras oncogene, in addition to that of the NIH 3T3 controls. In contrast, DNAs from the other two tumors did not contain any of the known oncogenes tested, except those endogenous to NIH 3T3 cells. DNAs from three of four first cycle tumorigenic transformants gave rise to morphologically transformed foci when assayed in a second cycle of transfection. DNAs from all three secondary transformants contained discrete human alu sequences, and in addition, contained Ha-ras sequences similar to those present in their respective primary transformants. Interestingly, DNA from both primary and secondary transformants of one particular human squamous cell carcinoma contained highly amplified copies of the Ha-ras oncogene. These results suggest that activation of the Ha-ras oncogene may be common in human skin cancers originating on sun-exposed body sites. Further characterization of the Ha-ras oncogenes present in these human skin cancers may provide information on the molecular mechanisms by which UV radiation of the sun induces human neoplasms on exposed body sites.     

Malignant conversion of murine squamous papilloma cell lines by transfection with the fos oncogene

Murine papilloma cell lines 308 and SP-1 have been used as recipients for transfected oncogenes to investigate malignant conversion. These cell lines express an activated c-rasHa gene with a codon 61 mutation and produce squamous papillomas when transplanted as skin grafts onto nude mice. They are not tumorigenic by subcutaneous injection. Both papilloma cell lines were stably transfected with plasmid DNA containing either a rearranged murine plasmacytoma-derived c-myc (minus exon 1), adenovirus 5 E1A, FBJ v-fos or a human c-fos/FBJ v-fos chimera, using cotransfection with the neomycin resistance gene contained in pSV2neo to select for transformants. Southern and northern blotting analysis confirmed the uptake and expression of exogenous DNA in both G418-selected cell lines and in the derived tumors. Unlike the E1A- and myc-containing plasmids, both fos constructs caused malignant conversion in either cell line, as defined by the squamous cell carcinoma histology of tumors from grafted cells and the development of carcinomas after subcutaneous injection into athymic nude mice. Immunofluorescence analysis for specific keratin gene expression indicated that tumors derived by introduction of either of the fos oncogenes were devoid of staining for K1, a 67 kDa epidermal keratin that is expressed in papillomas but not in squamous carcinomas. Tumors from E1A, myc, or pSV2neo transfectants expressed K1, although in a focal distribution. The malignant phenotype induced by the fos oncogene constructs was not associated with the ability to form agar colonies in vitro or to express gamma-glutamyl transpeptidase in the tumors. Since both 308 and SP-1 were sensitive to the fos oncogene for malignant conversion and insensitive to E1A or myc, it is possible that fos may cooperate with the endogenous-activated c-rasHa gene to convert these cells to malignancy. However, since gamma-glutamyl transpeptidase activity is found in the majority of chemically induced mouse skin carcinomas that possess an activated c-rasHa gene, fos activation may not be a common pathway for spontaneous malignant conversion.     

Transfer of a dominant-acting tumor-inducing oncogene from human prostatic carcinoma cells to cloned rat embryo fibroblast cells by DNA-transfection.

The mechanism by which normal human prostate cells develop into prostatic carcinoma cells is not presently known. In the present study we have tested the hypothesis that specific prostatic carcinomas develop as a consequence of activation of a cellular gene(s) with transforming and tumorigenic potential. To test this possibility, high molecular weight DNA was extracted from the human prostatic carcinoma cell line, LNCaP, and cotransfected with a dominant acting neomycin resistance gene, pSV2-neo, into a subclone of Fischer rat embryo fibroblast (CREF) cells, CREF-Trans 6, and NIH-3T3 cells. Cells were selected for growth in G418 and pooled resistant colonies, which were morphologically normal, were injected subcutaneously into athymic nude mice. Tumors developed in several of the animals inoculated with LNCaP DNA-transfected CREF-Trans 6 cells and they were established in monolayer culture. In contrast, no tumors developed in nude mice injected with untransfected CREF-Trans 6 cells, pSV2-neo transfected CREF-Trans 6 cells or LNCaP plus pSV2-neo DNA-transfected NIH-3T3 cells. DNA from the first cycle tumor-derived CREF-Trans 6 cell lines, which were morphologically transformed in monolayer culture, was cotransfected with pSV2-neo a second time into CREF-Trans 6 cells and transfected cells, which were still morphologically normal, were injected into nude mice. Tumors developed in animals and they were again established in tissue culture. Secondary transfectants isolated from animals were morphologically transformed and grew with high efficiency in agar. Both primary and secondary LNCaP-transfected-nude mouse tumor derived-CREF-Trans 6 cells contained human repetitive (Alu) sequences. Although the pattern of Alu integration in the tumor derived CREF-Trans 6 cells were different for different tumors, both primary and secondary tumors contained a single apparently common-sized Alu fragment. The present study indicates that the human prostatic carcinoma cell line, LNCaP, contains a dominant-acting tumor-inducing oncogene which does not induce morphological transformation of CREF-Trans 6 or NIH-3T3 cells in monolayer culture. In addition, the CREF-Trans 6 cell line can detect this tumor-inducing gene function, whereas this activity is not observed in DNA-transfected NIH-3T3 cells.     

Reconstituted basement membrane (matrigel) enhances the growth of human glioma cell lines in nude mice

Transplantation of human cancers into immunologically deficient mice is widely used to study potential therapeutic interventions in vivo. For brain tumor research, however, several factors limit more widespread application of this animal model. First, only a minority of human glioma-derived cell lines are tumorigenic in nude mice. In addition, even for tumorigenic cell lines, tumor take is variable and growth is often slow for tumors derived from cell inoculums. Reconstituted components of tumor basement membrane (matrigel) have been found to improve the growth in nude mice of several types of human tumors originating outside the central nervous system when premixed with the tumor cells before subcutaneous inoculation. We investigated the ability of matrigel to enhance the growth in nude mice of tumors derived from the human glioma cell lines U-251 MG, U-373 MG, SNB-78 and SNB-101.Athymic nude mice (NIH Swiss background, nu/nu genotype) were inoculated subcutaneously with 1.0 × 10⁶ tumor cells alone or after premixing with an equal volume of liquid matrigel. U-251 and U-373 cells were tumorigenic, with palpable tumors present by about 2 to 3 weeks. Co-injection of these cell lines with matrigel resulted in higher tumor-take rates, from 6/10 to 8/8 animals for U-251 at 60 days, and from 9/12 to 11/11 animals for U-373 at 60 days. Matrigel also enhanced tumor growth, with tumors at 45 days significantly larger than those formed in the absence of matrigel, for both cell lines (p < 0.01). SNB-78 and SNB-101 cells did not give rise to progressively enlarging solid tumors with or without matrigel.Matrigel enhances the growth of tumorigenic human gliomas in athymic nude mice. This technique provides a model with more consistent tumor take and more rapid growth kinetics for human glioma cell lines that are tumorigenic in nude mice.The U.S. Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged     

Malignant transformation of human bladder epithelial cells by DNA transfection with the v-raf oncogene

Transfection of the v-raf oncogene into immortalized, nontumorigenic human bladder epithelial cells resulted in the isolation of two tumorigenic transformants. Both were identified as human and of the same origin as the parent cell line by human leukocyte antigen typing and Southern blot analysis. Both the primary tumorigenic transfectants and the cell lines established from the induced tumors expressed v-raf mRNA and v-raf protein. In both tumorigenic transformants the level of c-myc mRNA was enhanced compared with that of the parent cell line.     

透明质酸酶(PH-20)对乳腺癌细胞MDA435生长的影响

目的　探讨人精子细胞膜结合型透明质酸酶 (PH 2 0 )基因对乳腺癌细胞MDA4 35在体内外生长的影响。方法　将PH 2 0cDNA转染到人乳腺癌细胞株MDA4 35中 ,形成MDA4 35 PH2 0。将MDA4 35 PH2 0种植在软琼脂培养皿中 ,以转染空载体pcD NA3的MDA4 35细胞为对照 ,观察PH2 0基因对乳腺癌细胞集落形成能力的影响。再将MDA4 35 PH2 0和MDA4 35 pcDNA3分别以同等条件接种到 5周龄裸鼠皮下 ,在不同时间点上比较两组鼠身上肿瘤大小。结果　转PH2 0基因的MDA4 35细胞在软琼脂培养基中集落形成数量明显高于对照组 ,同时MDA4 35 PH2 0在裸鼠身上的肿瘤生长速度也显著高于对照组。结论　PH 2 0使乳腺癌细胞MDA4 35的恶性度增高并促进其在体内的生长能力。     

Suppression of the tumorigenic growth of Burkitt's lymphoma cells in immunodeficient mice by cytokine gene transfer using EBV-derived episomal expression vectors

Epstein-Barr virus (EBV)-based expression vectors were tested for cytokine gene transfer-mediated induction of an immune response against human lymphoma cells. These vectors express the EBV latent gene EBNA 1 and carry the EBV latent origin of replication (ori P) for episomal replication in transfected cells. In addition, 3 human immunoglobulin light chain enhancer elements augment expression in B-cells. The suitability of these vectors for expression of cytokine genes in human lymphoma cells in vitro has been demonstrated. In order to extend these experiments in vivo, highly tumorigenic Burkitt's lymphoma (BL) cells were transfected with different cytokine genes of human and murine origin cloned into the EBNA 1/ori P vectors. Tumorigenicity of the transfectants was measured after inoculation into nude mice. No effect on tumorigenicity was observed after hIL 6 transfection and an inconsistent effect after hTNFalpha transfection. In contrast, complete suppression of tumor outgrowth occurred in hIL 10 transfectants. This tumor suppressive effect, however, was restricted to the IL 10 transfectants themselves and not directed against non-transfected cells. By comparison, mIL 4 transfected BL cells also were non-tumorigenic. However, co-inoculation of mIL 4 transfected and non transfected cells resulted in suppression of the tumorigenicity of the non-transfected cells. Thus, highly tumorigenic BL cells in nude mice are sensitive to immune effector mechanisms triggered by cytokine expression. In this experimental model, EBNA 1/ori P expression vectors are a suitable tool for cytokine gene transfer mediated induction of an anti-lymphoma immune response of the host.     

Site-directed mutagenesis of hamster complement C1S: Characterization with an active form-specific antibody and possible involvement of C1S in tumorigenicity

We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster CIs cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423 (CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. CIs produced from these transfectants lost their enzyme activity. Transfectants of these mutated CIs cDNA did not form tumors in nude mice. To distinguish between active and inactive CIs in situ, we have developed novel antibodies, one directed to the NH₂-terminal neoepitope of the L chain and the other specific for uncleaved inactive CIs. These antibodies were used to characterize CIs produced by transfectants, so as to determine whether or not it was cleaved at the right position. © 1996 Wiley-Liss, Inc.     

Selection for spontaneous metastasis after calcium phosphate-mediated transfer of DNA

The ras oncogene has been shown to induce metastatic potential in a wide variety of cells. However, one cell line, C127, when transformed by ras (HC127), proved to be an exception in that the transformed cells gained the ability to form tumors in nude mice, but these tumors did not form spontaneous metastases. Because C127 cells do not metastasize after ras transfection, these cells can be used to identify other factors which contribute to the development of metastatic potential. Specifically, these cells can be used as recipients in DNA transfer experiments utilizing DNA from other cells in which H-ras has been used to induce the metastatic phenotype, thus allowing the search for genes in addition to ras itself which may be necessary for metastasis. A system utilizing genomic DNA transfer into C127 cells transformed by ras has been developed. These cells were used as the recipient for genomic DNA in cotransfection with pSV2neo followed by selection both in the experimental i.v. assay and in the spontaneous metastasis assay in nude mice. DNA from two lines with metastatic potential (both transformed by ras genes) gave rise to metastatic clones, whereas DNA from the recipient cells, HC127, NIH 3T3 cells, and two human tumor cell lines, CHP126 and A2058, failed to give rise to transfectants which could metastasize. The metastases after the first cycle were put into culture, and DNA was extracted from these cells and used in a second cycle of transfection. The capacity to metastasize was also transferred in the second cycle. Of seven metastatic clones examined, four showed no detectable rearrangements of the transfected v-H-ras gene, but in three of these clones, there were rearrangements of this gene, which was originally present in the recipient cell, HC127. This indicates that in a subset of the selected clones there may have been selection for rearrangements of the host genome rather than introduction of foreign DNA sequences, and that such effects must be considered in gene transfer experiments. It is also possible that the transfer of particular genomic DNAs are leading to genetic instability in these experiments. mRNA levels were compared in the metastatic variants and the parental cells; H-ras-specific RNA was raised from 4- to 22-fold in the metastatic cell lines, regardless of rearrangement of the v-H-ras gene.     

Vascularity, perfusion rate and local tissue oxygenation of tumors derived from ras-transformed fibroblasts.

Tumors derived from ras-transformed rat fibroblasts were investigated in order to gain insight into possible interrelationships between oncogenic transformations and therapeutically relevant parameters of the metabolic micromilieu of solid tumors in vivo. Tumors grew in nude mice after injection of in vitro-passaged cells. Growth rates, early stages of angiogenesis, perfusion and tissue oxygenation were assessed. Compared with the parental cell line, both ras transformants grew very rapidly and exhibited an early onset of angiogenesis. Perfusion rates of one ras-transformed tumor line were similar to those of the parental tumors whereas reduced flow values were detected in tumors of the other ras line. The better perfused tumors exhibited more adequate tissue oxygen levels whereas reduced oxygen levels were obvious in the poorly perfused ras line. These results indicate that ras transformation alters therapeutically relevant parameters of the tumor micromilieu. However, the tumor phenotype cannot be predicted even if the transforming oncogene is known.