Morphological and functional differentiation of Kirsten murine sarcoma virus-transformed rat adrenocortical cell lines

This study was undertaken to examine relationships of the phenotype of malignant cells to target cell properties and to events early in the transformation process. Eighteen transformed lines were obtained by Kirsten murine sarcoma virus infection of cells from adrenal glands of rats ages 4 to 30 weeks, at first or second passages in culture. They were grown either as fibroblastic adrenocortical stem cells or as more differentiated epithelial cells, depending on culture conditions. Of 14 lines examined for their capacity to synthesize corticosteroids, 11 converted [14C]pregnenolone to progesterone, and one converted to deoxycorticosterone. In vivo, seven lines produced tumors resembling pleomorphic carcinomas, six lines grew as sarcomas, four grew as mixed tumors, and one line produced anaplastic tumors. Distinguishing features in culture of the carcinoma-producing lines were early onset and rapid progression of morphological transformation, a noncohesive epithelial cell form in some lines, lack of extracellular matrix, and, possibly, and origin in older animals. In contrast, sarcoma-producing cells were fibroblastic and cohesive, produced extracellular matrix, and transformed morphologically after longer and less well-defined periods in culture. The variation in histopathology was unrelated to the differentiation of the target cells and to the capacity of the transformed cells to synthesize corticosteroids. The results show that adrenocortical cells, transformed by Kirsten murine sarcoma virus after short-term culture, usually retain some functional differentiation and sometimes resemble human adrenocortical carcinomas histologically. The susceptibility of adrenocortical cells to Kirsten murine sarcoma virus raises the possibility that mesodermally derived epithelia in general may be target tissues for C-type sarcoma viruses.     

Normal Human Chromosome 1 Carries Suppressor Activity for Various Phenotypes of a Kirsten Murine Sarcoma Virus-transformed NIH/3T3 Cell Line

In order to identify chromosomes that carry putative tumor-suppressor genes for the various phenotypes of Kirsten sarcoma virus-transformed NIH/3T3 (DT) cells, we performed microcell-mediated chromosome transfer into DT cells. We first isolated mouse A9 clones, containing a single human chromosome 1, 11 or 12 tagged with pSV2- neo plasmid DNA. Then, chromosome 1, 11 or 12 was transferred from the A9 clones into DT cells by microcell fusion. The growth rate, colony-forming ability in soft agar and tumorigenicity of the DT cells were controlled by chromosome 1, but not by chromosome 11 or 12, indicating that normal human chromosome 1 carries a putative tumor-suppressor gene(s) that affects various transformed phenotypes of DT cells.     

Antigenic phenotype of NIH 3T3 cell line

The antigenic phenotype of the NIH 3T3 cell line was examined by use of a panel of monoclonal antibodies and alloantisera specific for H-2 and several non-H-2 antigens. The binding of antibodies to cell surface antigens was examined by a complement-dependent microcytotoxicity test and indirect immunofluorescence quantitated by flow cytometry. The phenotype of the tested NIH 3T3 cell line was H-2q, Qa-2, Ly-6.2, Thy-1.2, Ly-23.2, and 9F 3+. The expression of H-2 antigens (Kq, Dq/Lq) was lower than that in the T-lymphocytes of the B10.Q (H-2q) strain, and the expression of Qa-2 was very low. From the Ly-6 complex, the antigen Ly-m6.2A was strongly expressed, while Ly-m.6B, Ly-m.6C, and antigens ThB and H9/25 were not detected. A monoclonal antibody specific for the nonpolymorphic antigen 9F 3 brightly stained 100% of NIH 3T3 cells. Inasmuch as the NIH 3T3 immortalized cell line was developed from outbred NIH Swiss mice, a syngeneic recipient for this cell line does not exist. However, on the basis of the determined antigenic phenotype the host most compatible to NIH 3T3 cells can be selected for in vivo experiments, where an immunocompetent recipient is required. Two inbred mouse strains identical with NIH 3T3 cells in the antigens examined in this study, B10.Q and DBA/1, are of potential use for transplantation with NIH 3T3 cells.     

Transformation of NIH/3T3 cells by DNA from a human hepatoma cell line with integrated hepatitis B virus DNA

We have studied by means of DNA-mediated gene transfer the transforming activity of the DNA of the human hepatoma cell line HCC-M, which contains genomes of hepatitis B virus (HBV) in integrated form. DNA from HCC-M induced transformed foci on transfection of NIH/3T3 cells. DNAs from primary transformants were capable of inducing secondary transformants. Most of the DNAs of these transformants were demonstrated to contain both human repetitive sequences and HBV DNA, indicating that the transformants had incorporated exogenous human DNA and HBV DNA as well. These results suggest that transformation occurs as the result of the transfer of oncogene which might be closely associated with HBV genome.     

Normal human chromosome 11 suppresses tumorigenicity of human cervical tumor cell line SiHa

We examined the ability of human chromosome 11 derived from normal fibroblast cells to suppress the tumorigenicity of SiHa cells, a human cervical tumor cell line. Using DNA transfection, the human chromosome was tagged with a selectable marker (the pSV2neo gene, which encodes resistance to the antibiotic G418), transferred to mouse A9 cells by cell hybridization and microcell transfer techniques, and then transferred to SiHa cells by microcell transfer. These procedures resulted in the appearance of 15 independent, G418-resistant clones, 5 of which had one or two extra copies of an intact human chromosome 11. In situ chromosomal hybridization of these clones with the pSV2neo plasmid revealed the presence of a neo-tagged human chromosome 11 in all of the five SiHa-microcell hybrids. Two SiHa-microcell hybrids that contained a single copy of neo-tagged human chromosome 12 were also isolated by the same methods. The tumorigenicities of SiHa clones with one or two extra copies of chromosome 11 (SiHa-11) were suppressed; four of the five SiHa-11 clones formed no tumors in nude mice, whereas both parental SiHa cells and SiHa cells with an extra chromosome 12 formed tumors within 30 d. One SiHa-11 cell clone formed a single tumor 90 d after injection. This rare tumor had lost one copy of chromosome 11 and rapidly formed tumors when reinjected. These results indicate that the introduction of a single copy of normal human chromosome 11, but not chromosome 12, suppresses the tumorigenicity of SiHa cells, indicating the presence on human chromosome 11 of a putative tumor-suppressor gene (or genes) for human cervical tumors.     

Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance.

Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro. However, the relevance of these to tumor cell resistance in vivo remains unclear. We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy. One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522. The sensitivities of six other sarcoma lines were similar to that of AG1522. STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment. Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations. Accumulation of [3H]daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil. Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher. This was due mostly to an increase in selenium-dependent peroxidase activity. After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry. Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine. These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.     

Multiple chromosomes carrying tumor suppressor activity for a uterine endometrial carcinoma cell line identified by microcell-mediated chromosome transfer

Putative tumor suppressor genes can be mapped to specific chromosomes by the introduction of individual chromosomes derived from normal cells via microcell fusion. We have examined whether a highly malignant human uterine endometrial carcinoma cell line, HHUA, can be suppressed by only one normal chromosome or by multiple chromosomes. A library of mouse A9 clones containing different human chromosomes tagged with the pSV2-neo plasmid DNA were constructed. Transfer by microcell fusion of either chromosome 1, 6, 9, 11, or 19 into the HHUA tumor cell line was performed, and the abilities of the microcell hybrids to form tumors in nude mice were examined. The introduction of a chromosome 19 had no effect on the tumorigenicity of the cells, whereas microcell-hybrid clones with an introduced chromosome 1, 6 or 9 were completely suppressed for tumorigenicity. A decrease in tumor-take incidence in some but not all clones was observed following the introduction of a chromosome 11. The nontumorigenic microcell hybrids with an introduced chromosome 1 differed from the nontumorigenic microcell hybrids with an introduced chromosome 6, 9, or 11. A large percentage of hybrids with chromosome 1 senesced and/or showed alterations in cellular morphology and transformed growth properties in vitro. No growth or morphology alterations were observed following transfer of the other chromosomes. These results may indicate that more than one chromosome carries a tumor suppressor gene(s) for this human uterine endometrial carcinoma cell line and support the hypothesis that multiple tumor suppressor genes control the tumorigenic phenotype in the multistep process of neoplastic development.     

Proliferation and tumorigenesis of a murine sarcoma cell line in the absence of DICER1

MicroRNAs are a class of short ~22 nucleotide RNAs predicted to regulate nearly half of all protein coding genes, including many involved in basal cellular processes and organismal development. Although a global reduction in miRNAs is commonly observed in various human tumors, complete loss has not been documented, suggesting an essential function for miRNAs in tumorigenesis. Here we present the finding that transformed or immortalized Dicer1 null somatic cells can be isolated readily in vitro, maintain the characteristics of DICER1-expressing controls and remain stably proliferative. Furthermore, Dicer1 null cells from a sarcoma cell line, though depleted of miRNAs, are competent for tumor formation. Hence, miRNA levels in cancer may be maintained in vivo by a complex stabilizing selection in the intratumoral environment.     

A mutant cell line derived from NIH/3T3 cells: two oncogenes required for in vitro transformation

EK-3, a cell line derived from NIH/3T3 cells, was isolated. These cells are nontumorigenic to NIH Swiss nude mice. They required both myc and ras genes for in vitro transformation in contrast to NIH/3T3 cells, which are efficiently transformed following transfection by ras alone. Two other genes, chloramphenicol acetyl transferase and geneticin resistance, could be efficiently transfected and expressed in both EK-3 cells and the parental NIH/3T3 cells. Thus the possibility that the requirement of myc in EK3 cells is due to low efficiency of transfection could be ruled out. The present study suggests that myc plays a significant role in the overall process of transformation rather than simply immortalization of the cells. The EK-3 line can be very helpful in elucidating this function.     

Effect of adeno-associated virus on transformation of NIH 3T3 cells by ras gene and on tumorigenicity of an NIH 3T3 transformed cell line

Transfection of NIH-3T3 cells with the plasmid pJ234, containing DNA from the human bladder carcinoma T24 cell line (ras gene), results in their transformation. Adeno-associated virus did not affect significantly the number of the transformed foci when different multiplicities of infection were used and when the virus was added to the cultures at different time intervals before or after transfection. A transformed cell line was derived following transfection of NIH 3T3 cells by the ras gene. Infection of these cells with adeno-associated virus resulted in a decrease in their growth rate and cloning efficiency. These infected cells showed a dose-dependent reduction in the frequency and an increase in the latent period for tumor appearance in nude mice.     

Antiproliferative effect of interleukin-1 on human ovarian carcinoma cell line (NIH:OVCAR-3).

The human ovarian carcinoma cell line, NIH:OVCAR-3, possesses high affinity receptors for interleukin-1 (IL-1). Binding experiments with 125I-IL-1 alpha indicate a dissociation constant of approximately 55 pM and the presence of approximately 7800 receptors/cell. These receptors bind both IL-1 alpha and IL-1 beta and internalize IL-1. Proliferation is NIH:OVCAR-3 cells is inhibited by IL-1. Half-maximal inhibition is observed with 2-3 units/ml of IL-1 alpha or IL-1 beta. A maximal effect (80% inhibition of cell proliferation) is achieved by treatment of cells with greater than or equal to 10 units/ml of IL-1 for 3 days. The antiproliferative effect of IL-1 is blocked by IL-1 receptor antagonist. Light and electron microscopy studies show that IL-1 treatment causes cytopathological changes and a reduction in the number of mitotic figures in NIH:OVCAR-3. IL-1 stimulates prostaglandin E2 release by NIH:OVCAR-3 cells, but this response is unrelated to the antiproliferative effect of IL-1. Interferon-alpha A (IFN-alpha A) also inhibits growth of NIH:OVCAR-3 cells in a concentration-dependent manner. Combination of IFN-alpha A and IL-1 gives synergistic inhibition of NIH:OVCAR-3 cell proliferation. IL-1 alone or in combination with IFN-alpha A or other agents may be useful for treatment of human ovarian cancer.     

BRCA1 carries tumor suppressor activity distinct from that of p53 and p21.

The loss of BRCA1 function appears as an essential step in breast and ovarian epithelial cells oncogenesis and is consistent with the concept that BRCA1 acts as a tumor suppressor gene. However, the mechanism underlying this activity is not understood. In 1996, a retroviral vector was used for BRCA1 delivery to demonstrate that the transfer of BRCA1 inhibits breast and ovarian cancer cell growth. Since this early observation, the tumor growth inhibitory activity of BRCA1 in vivo has not been further documented. Here we re-address this issue and report experiments designed to evaluate the potential of adenovirus-mediated BRCA1 delivery to suppress the growth of cells with various status of endogenous BRCA1 in comparison with p53 and p21. Delivery of wild-type BRCA1 by an adenovirus vector in breast and ovarian tumor cells, decreases in vitro proliferation and tumorigenicity. Similarly, in vivo administration of BRCA1 provokes tumor growth retardation or regression comparable to that obtained with p53 or p21. The antitumor effect of BRCA1 is not observed upon transfer of a mutant lacking the 542 C-terminal residues. The p53- or p21-mediated antiproliferative activities are likely to bear on their capacity to induce apoptosis and/or interfere with cell cycle checkpoint. By contrast, the data presented here show that neither of these mechanisms can account for the BRCA1-mediated antitumor activity and suggest the activation of an alternative route.     

Establishment and characteristics of a human synovial sarcoma cell line

A human synovial sarcoma cell line (HSS-84), from a painful mass excised locally near the left sternoclavicular joint, was established and has been continuously propagated in vitro for more than one year through 54 passages till the end of 1985. Doubling time was 65 hr. The index of mitosis was 28.8%. The chromosome number for most of the cells was hypertriploid. The cells, stored in liquid nitrogen, had a good revival. Cytologically, some cells, showing elongated, spindle, stellate or spidery shape with prominent processes, were arranged in loose or bundled pattern, similar to sarcoma cells; some cells showed ovoid and polygonal in mosaic arrangement, similar to epithelioid cells. Both features indicated biphasic differentiation of the cells. By electron microscopy, the cells consisted of light and dark cells with microvilli and obvious abnormality. Histopathologically, the tumor, from which HSS-84 originated, was a biphasic differentiation synovial sarcoma. It was composed of malignant spindle interstitial cells and malignant epithelioid cells lining the clefts or spaces. The latter was similar to cultured cells by electron microscopy. HSS-84 was identical with the donor. HSS-84 establishment will be useful for research on diagnosis, therapy, origin and biphasic differentiation of the synovial sarcoma.