Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.     

Establishment and characterization of human signet ring cell gastric carcinoma cell lines with amplification of the c-myc oncogene.

Two unique human signet ring cell gastric carcinoma cell lines (designated HSC-39 and HSC-40A) were established in vitro from the ascites of a 54-year-old male patient. Both cell lines were biologically quite similar, grew in vitro in suspension with a population doubling time of 28-30 h, and had cytological features of mucinous epithelial tumor cells. They formed colonies in soft agar, with a cloning efficiency of 0.8-1.0%. Ultrastructurally, numerous granules were observed in the cytoplasm, suggesting secretory activity. The frequent presence of desmosome and the tight junction at the cell boundary certifies the epithelial origin of the lines. Immunocytochemistry and radioimmunoassay showed production of tumor marker antigens (carcinoembryonic antigen, CA 19-9, and sialyl-Lex-i) and gastrin in both lines. These lines were transplantable in athymic BALB/c nude mice. The histopathology of each line growing in athymic BALB/c nude mice was similar to that of the original tumor. The karyotype of the cells was highly aberrant with structural and numerical changes. The presence of numerous double minute chromosomes and loss of the 13 chromosome and Y-chromosome characterize these lines. In addition, the amplified c-myc oncogene (16-32-fold) was found in both cell lines and original ascitic tumor cells. Overexpression of the c-myc mRNA was noted. These cell lines may be a useful tool, providing both in vivo and in vitro systems for further studies of the biology and therapy of human signet ring cell (or Borrmann's type IV carcinoma) gastric carcinoma.     

Comparison of the growth and metastasis of four human intestinal tumor cell line xenografts

The growth and metastasis of four human intestinal tumor cell lines: one duodenal adenocarcinoma (HTB-40) and three adenocarcinomas of the colon (CCL-218, CCL-222 and HT-29) have been compared in vitro and in vivo in nude mice. HTB-40 was the fastest growing cell line in vitro with a doubling time (DT) of 14.8 h. CCL-218 and CCL-222 grew more slowly in vitro with doubling times of 21.6 and 22.8 hours, respectively. All three of these tumors grew more slowly in vivo with doubling times ranging from 39.1 h (CCL-218 in male nude mice) to 65.3 h (CCL-222). The growth of CCL-218 cells was significantly slower in female nude mice DT 51.0 h). HT-29 was the slowest growing in vitro (DT 23.8 h) and in vivo (DT about 100 h). HT-29 also showed the greatest discrepancy between its DT measured in vivo as compared to in vitro, suggesting a greater clonogenic cell loss from HT-29 tumors in vivo. Histologic evaluation of these tumors grown subcutaneously in nude mice showed all to be anaplastic and to produce liver micrometastases. However, more extensive abdominal and liver metastases were observed in the nude mice injected with HT-29 cells, and some of these metastases had morphologic features of moderately well-differentiated epithelium. These results indicate the usefulness of the HT-29 tumor cell line as an experimental model of metastasis from a human colonic adenocarcinoma.     

Lack of comparability between binding of monoclonal antibodies to melanoma cells in vitro and localization in vivo

The affinity and specificity of monoclonal antibodies (MAbs) to tumor-associated antigens are often determined by in vitro assays. The specific binding of two anti-human melanoma antibodies (96.5 and ZME-018) and a control antibody (ZCE-025) to three human melanoma cell lines (DX3, A375-M, and Hs294t) was examined under in vitro conditions and compared to in vivo localization of 111In-labeled antibodies to the same cells growing as solid tumors in the subcutis of nude mice. The in vitro binding of the specific MAbs to the tumor cells did not predict in vivo localization. Under in vitro conditions, MAb ZME-018 bound to all three cell lines at levels exceeding that of 96.5, yet ZME-018 did not show superior localization to subcutaneous tumors. MAb 96.5 bound to cultured DX3 cells at levels exceeding those observed with A375-M cells. Yet, 96.5 localized better to A375-M xenografts in nude mice than to DX3 or Hs294t xenografts. Antigen expression differed between in vitro and in vivo growing cells, as evidenced by alteration in binding of 96.5 to tumor cells dissociated from solid subcutaneous tumors. Collectively, the data suggest that in vitro parameters do not predict the clinically relevant localization of MAbs to tumors.     

重组腺病毒介导反义c-myc基因对人肝癌细胞系的治疗作用

目的：探讨重组腺病毒介导反义ｃ－ｍｙｃ基因（Ａｄ－ＡＳｍｙｃ）治疗人肝癌细胞的作用。方法：观察Ａｄ－ＡＳｍｙｃ对人肝癌细胞系的转导效率,通过细胞生长曲线、克隆形成实验、ＤＮＡ片段化分析、ＲＴ－ＰＣＲ、裸鼠皮下移植瘤治疗实验,分析Ａｄ－ＡＳｍｙｃ对人肝癌细胞系Ｂｅｌ－７４０２、ＱＳＧ－７７０１、ＳＭＭＣ－７７２１和ＨＣＣ－９２０４细胞生长和ｃ－ｍｙｃ基因表达及裸鼠肿瘤生长的抑制作用。结果：Ａｄ－ＡＳｍｙｃ可高效转导人肝癌细胞系,抑制细胞生长;转染细胞克隆形成能力降低,克隆成活率为对照组的５３．９％～６９．１％,ｃ－ｍｙｃ基因表达下降;Ａｄ－ＡＳｍｙｃ处理肝癌细胞,ＤＮＡ凝胶电泳出现明显的梯形条带,瘤内注射Ａｄ－ＡＳｍｙｃ可抑制裸鼠皮下移植瘤生长。结论：重组腺病毒介导的反义ｃ－ｍｙｃ基因转移,有可能成为肝癌基因治疗     

Role of polyamines in the growth of hormone-responsive and -resistant human breast cancer cells in nude mice.

Recent in vitro data suggest that at least some hormone-independent breast cancer cells exhibit increased polyamine biosynthesis and resistance to antipolyamine therapy. To address this issue under conditions of in vivo growth, we tested the antiproliferative effect of the polyamine synthetic inhibitor alpha-difluoromethyl-ornithine (DFMO) on hormone-dependent (MCF-7) and -independent (MDA-MB-231, BT-20) breast cancer cell lines growing in nude mice. We observed that DFMO significantly inhibited the growth of established tumors to a similar extent in all cell lines, even though tumor regression was only observed with MCF-7 cells. DFMO, while inhibiting E2-supported MCF-7 breast cancer growth, did not inhibit E2-stimulated progesterone receptor synthesis. Cellular levels of polyamines were highest in MCF-7 cells and lowest in the BT-20 cell line. Tumor content of spermidine was similarly suppressed by DFMO treatment in the 3 cell lines, while the spermine level was unaffected. Cellular putrescine levels were suppressed in MCF-7 and BT-20 cells. Administration of DFMO prior to implantation of fragments of MCF-7 or MDA-MB-231 tumors in nude mice significantly inhibited tumor development to a similar extent. The action of DFMO seemed to be predominantly tumoristatic since new tumors develop in some mice upon discontinuation of the drug. We conclude that the hormone-independent breast cancer cell lines tested do not exhibit increased polyamine biosynthesis or resistance to antipolyamine therapy when grown in vivo in nude mice.     

Bi-modal differentiation pattern in a new human neuroblastoma cell line in vitro.

We have isolated a human neuroblastoma (NB) cell line, HTLA230, from the bone-marrow aspirate of a patient with stage-IV disease. Subcutaneous tumors after inoculation of HTLA230 cells into nude mice were composed of primitive neuroblasts which rarely contained neuro-secretory granules. Cytogenetic studies of the cell line demonstrated 2 distinct populations of cells with common chromosomal markers. Stable sub-clones with a differentiated or undifferentiated cell morphology were isolated, demonstrating phenotypical heterogeneity of the HTLA230 parental cell line. Treatment with retinoic acid (RA) induced extensive neurite outgrowth in the parental cell line and in phenotypically differentiated sub-clones, but rarely in undifferentiated ones. Long-term treatment with RA was not associated with down-modulation of mycN-gene expression, which could be achieved only in cultures treated additionally with aphidicolin, a DNA-synthesis inhibitor, thus eliminating growing NB cells. A RA resistant subclone (CI-5) was isolated from parental HTLA230 cells grown at clonal cell density. Cells originally showed a homogeneously differentiated morphology; however, flat cells (F-cells) appeared with time and were subsequently separately propagated. Transdifferentiation of isolated F-cells into cells with neuron-like (N-cell) morphology was observed. Immunohistochemical analysis demonstrated that F-cells had lost the expression of neuronal markers, including HNK-I and A2B5, and expressed the intermediate filament, vimentin. Furthermore, F-cells showed high incorporation of [methyl-3H] thymidine (3H-TdR) by autoradiography but no mycN protein could be detected, although present in the parental cell line. These results then suggest that the isolated NB cell line and the RA-resistant variant line represent an excellent in vitro model with which the bi-modal differentiation pathway of NB can be analyzed on a molecular biological level.     

Biology, cytogenetics, and sensitivity to immunological effector cells of new head and neck squamous cell carcinoma lines

Twenty-one head and neck squamous cell carcinoma (HNSCC) cell lines were established from 89 fresh tumor specimens in order to study the biology of HNSCC lines, establish tumors in nude mice, and evaluate the sensitivity to immunological effector cells of these tumors in vitro and in vivo in nude mice. The lines were established from explants using differential trypsinization and culture for 2 to 20 mo. The explants were derived from 11 different sites. Three pairs of lines were derived from both the primary tumor and metastatic lymph nodes in the same patients. All cultures grew as either compact or diffuse adherent monolayers, and they had a median doubling time of 86 h (range, 33 to 531 h). DNA fingerprinting confirmed that the HNSCC lines were individual isolates. Thirteen of 14 lines tested induced tumors in athymic mice. The histology of each line growing in nude mice was similar to that of the original tumor tissue. Immunocytochemistry showed keratin production in all lines tested. Aneuploidy (36 to 87 chromosomes) was present in all 16 lines studied; the median chromosome number for lines derived from primary tumors was 70, whereas for lines originating from metastatic or recurrent tumors, it was 54. Karyotypic analysis showed deletion of the short arm of chromosome 3 (3p-) in 12 of 16 cell lines and trisomy 6 in 12 of 16 lines. In addition, translocations between chromosomes 9 and 11 or 9 and 12 were each present in five of 16 lines tested. The HNSCC lines were resistant to lysis by natural killer cells, but were efficiently lysed by lymphokine-activated killer cells in 4-h 51Cr release assays. These new lines have allowed us to establish a model of local adoptive immunotherapy of HNSCC in tumor-bearing nude mice, and they provide a resource for future studies of the biology of HNSCC.     

Episome generated C-myc antisense RNA inhibits growth and tumorigenicity of a human neuroendocrine tumor-cell line

The neuroepithelioma cell line CHP100 expresses low but detectable amounts of N-myc protein together with large amounts of c-myc protein. We have recently demonstrated that antisense inhibition of N-myc expression in CHP100 cells leads to decreased in vitro growth and alterations in cellular morphology without affecting tumorigenicity in nude mice. In this study we report the construction of an episomally replicating vector designed to generate RNA antisense to part of the human c-myc gene. Such a Vector is able to inhibit c-myc expression in cell lines carrying multiple copies of the gene. Inhibition of c-myc expression leads to a decrease of in vitro growth and cloning efficiency and in vivo tumorigenicity of CHP100 cells. Our findings suggest that N-myc and c-myc subserve different functions in regulating the biology of CHP100 cells.     

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.     

Oncoprotein (c-myc, c-erbB1, c-erbB2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung. Clinical and biological correlations.

The expression of the protooncogene encoded proteins (c-erbB1, c-erb B2, c-myc, c-fos) and the suppressor gene product p53 was analyzed in 81 human squamous cell carcinomas of the lung and correlated with clinical parameters of the patients (patient survival, presence of metastases and tumor stage) and with biological characteristics of the tumors (tumor growth in nude mice, DNA-ploidy, proliferative activity, drug-resistance and P-glycoprotein or gluathione S-transferase expression). By means of immunohistochemistry, expression of c-erbB1 oncoprotein (EGF-receptor) was detected in 79% of the tumors, c-erbB2 (c-neu) proteins in 35%, c-myc proteins in 48%, c-fos proteins in 41%, and p53 in 43% of the tumors. Patients with c-erbB1 positive tumors had a poor prognosis (p = 0.021). In addition, these tumors were more frequently drug resistant (p = 0.0067). A significant correlation between the growth of the squamous lung carcinomas in nude mice and c-fos oncoprotein expression was demonstrated (p = 0.017). Therefore, EGF-receptor and c-fos products may serve as prognostic factors for the aggressiveness of squamous cell carcinomas of the lung and for the response of these tumors to chemotherapy. No significant correlation was found between the expression of the c-erbB1 or c-fos gene products and stage, metastasis and DNA-ploidy. In contrast to these results, no relationship was found between c-neu or c-myc gene products expression and any of the clinical or biological parameters examined. Aneuploid squamous cell carcinomas of the lung expressed p53 more frequently than diploid tumors (p = 0.027). However, there was no significant difference between p53 expression and stage, survival of patients, metastasis, growth of the tumors in nude mice, proliferative activity and drug-resistance of the tumors.     

Osteopontin is required for full expression of the transformed phenotype by the ras oncogene

The secreted phosphoprotein osteopontin (OPN) is strongly associated with the process of neoplastic transformation, based both on its pattern of expression in vivo and in vitro and on functional analyses. We have used 3T3 cells derived from wildtype and OPN-deficient mice and transformed by transfection with oncogenic ras to assess the role of OPN in transformation in vitro and in tumorigenesis in vivo. There was no effect of an absence of OPN on the ability of the cells to undergo immortalization or to form morphologically transformed foci following ras transfection. Wildtype and OPN-deficient cell lines were established from such foci, and lines with similar ras mRNA levels selected for further analysis. Ras-transformed cell lines from both wildtype and OPN-deficient mice could form colonies in soft agar indicating that this process can occur in the absence of OPN. However, the ability of the OPN-deficient cell lines to form colonies was reduced as compared to wildtype cell lines. Tumorigenesis in syngeneic and nude mice was assessed for a subset of cell lines that formed colonies efficiently in soft agar. Cell lines unable to make OPN formed tumors in these mice much more slowly than wildtype cells, despite similar growth of the cells on plastic and in soft agar. Taken together, these results indicate that maximal transformation by ras requires OPN expression, and implicate increased OPN expression as an important effector of the transforming activity of the ras oncogene.     

Efficient induction of focus formation in a subclone of NIH3T3 cells by c-myc and its inhibition by serum and by growth factors

In both experimental and spontaneous tumors, c-myc expression is often enhanced following its amplification or its rearrangement adjacent to a strong promotor/enhancer. However, c-myc by itself does not induce foci efficiently in fibroblast cultures. The effect of high levels of c-myc expression from a retroviral construct was investigated in several rodent fibroblast cell lines grown in medium containing 10% fetal calf serum or in serum-free PC-1 medium. c-myc-infected NIH3T3 clone 7 cells exhibited efficient quantitative focus formation when grown in PC-1 medium, whereas foci were not detected when grown in serum-supplemented medium. NIH3T3 clone 7 was the only cell line found to be sensitive to c-myc; other clones of NIH3T3 or other rodent fibroblast cell lines proved to be resistant to c-myc focus formation. At least two major types of morphologically distinct c-myc-induced foci were observed; the first was similar to ras-transformed foci induced in NIH3T3 and other fibroblast cell lines, and the second type was composed of adipocyte-like cells similar to NIH3T3 L1 cells. The c-myc infected cells cloned from these two types of foci expressed high levels of retrovirus-derived c-myc RNA and exhibited elevated levels of immunoreactive myc protein, as detected by immunofluorescent staining with an anti-myc polyclonal antibody. c-myc-transformed clones displayed only a limited ability to grow in soft-agar in the presence of serum and were not tumorigenic in nude mice. Focus formation by c-myc was quantitatively inhibited by the addition of interferon alpha + beta (INF alpha, beta), tumor necrosis factor alpha (TNF alpha) or transforming growth factor beta 1 (TGF beta 1) to the serum-free PC-1 medium, and, in correlation, NIH3T3 clone 7 cells produced the lowest level of endogenous TGF beta of the various cell lines tested.     

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.     

Malignant transformation of host cells by a human small cell lung cancer xenografted into nude mice

Malignant transformation of mouse host cells by a human small cell lung cancer (SCLC) was demonstrated by short-term in vitro cultivation of the tumor cells from a xenograft at two different transplant generations. Isoenzyme (LDH) and chromosome analysis showed that out of the 3 cell lines established from this tumor, 1 retained a human karyotype similar to that of the xenograft and 2 were murine transformed cell lines. These murine cell lines produced fibro-sarcoma-like tumors when injected into nude mice. Because of the early in vitro emergence of murine transformed cell populations, it is likely that the transformation process had occurred in vivo. Since in our experience the induction of transformation of host murine cells, also observed directly in vivo, is more frequent with SCLC than other histotypes (lung and colorectal adenocarcinoma), it is suggested that the known production of growth factor by these tumors may contribute to this transformation.     

博安霉素对人食管癌等抑瘤作用的实验研究

目的：研究博安霉素（boanmycin)在体内外对人食管癌细胞等的抑瘤作用。方法：采用MTT法检测博安霉素在人体外对5种人癌细胞的细胞毒作用。同时用裸鼠异种移植人食管癌细胞模型观察博安霉素对人食管癌的抑瘤作用。结果：博安霉素在体外对5种肿瘤细胞均有明显的细胞毒作用。博安霉素在10．0mg/kg和15．0mg/kg剂量下对裸鼠异种移植人食管癌HEC2的抑制率分别为85．2％，90．6％；在7．5mg/kg和15．0mg/kg剂量下对裸鼠异种移植人食管癌Eca-109的抑制率分别为81．2％，92．0％，结论：博安霉素在体内外对人食管癌细胞等具有显著的抑瘤作用。