Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.     

Enhancement of the invasive ability of a transformed human bronchial epithelial cell line by 12-O-tetradecanoyl-phorbol-13-acetate and diacylglycerol

The effect of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was studied using an immortalized human bronchial epithelial cell line, BEAS-2B, both in vivo and in vitro. The in vivo model consisted of tracheas reconstituted with an epithelium of BEAS-2B cells xenotransplanted into athymic nude mice. Intraluminal TPA treatment caused increased BEAS-2B cell proliferation and downgrowth into the tracheal stroma. In an in vitro invasion assay, TPA enhanced the invasive capacity of BEAS-2B cells 20- to 25-fold. A similar result was observed with diacylglycerol (DAG), an endogenous activator of protein kinase C, and the effects of TPA and DAG were abolished by simultaneous treatment with H-7, a protein kinase C inhibitor. TPA induced type IV collagenolysis, and this effect also was prevented by H-7. These data are consistent with the hypothesis that TPA causes these cells to become invasive by inducing collagenase activity and that this effect is mediated via protein kinase C.     

Suppression of tumorigenicity of a human lung carcinoma line by nontumorigenic bronchial epithelial cells in somatic cell hybrids

Hybrid cell lines between HuT292-DM, a human lung carcinoma line resistant to 6-thioguanine and ouabain, and either normal human bronchial epithelial cells (NHBE) or an SV40 "immortalized" but nontumorigenic derivative thereof (BEAS-2B), have been isolated by double selection. Hybrids of NHBE and HuT292-DM cells senesced after 40-43 population doublings in culture. In contrast, hybrids of BEAS-2B and HuT292-DM showed no sign of a culture "crisis" and have an indefinite life span. HuT292-DM cells produced tumors in 100% of athymic nude mice with a mean latency of 27 days, whereas tumorigenicity was totally suppressed in 76% of the BEAS-2B x HuT292-DM hybrids, with a 2- to 3-fold increased tumor latency in the remaining 24% of these hybrids. While the hybrids are hypotriploid to hypotetraploid, the parental lines are hypodiploid. The growth of HuT292-DM cells is stimulated, whereas NHBE and BEAS-2B cells are inhibited by serum. The growth response of the BEAS-2B x HuT292-DM hybrids to serum is similar to that of HuT292-DM cells. Thus, tumorigenicity and culture longevity are dominantly controlled by the nontumorigenic parent (NHBE or BEAS-2B). On the other hand, serum responsiveness is more similar to that of the tumorigenic parent (HuT292-DM).     

Altered p53 gene structure and expression in human epithelial cells after exposure to nickel.

The carcinogenicity of certain nickel compounds is well known. We have previously shown that human kidney epithelial cells were immortalized by treatment with Ni(II) and in cooperation with the v-Ha-ras oncogene transformed the cells to acquire tumorigenicity in athymic nude mice. Immunocytochemistry and sequence analysis of DNA from the nickel-immortalized cells revealed abnormal p53 expression and a T----C transition mutation in codon 238. These data are consistent with the hypothesis that Ni(II)-induced mutation in the p53 gene can be involved in the escape from senescence of kidney epithelial cells.     

Clonal variation of tumorigenic potential in v-Ha-ras-transformed human bronchial epithelial cells: Relationship to ras oncogene expression and CAD gene amplification

Infection of an SV40 large-T antigen-“immortalized” human bronchial epithelial cell line with a Zip-v-Ha-ras retroviral vector resulted in a mass culture that was tumorigenic in athymic nude mice. A tumor cell line derived from passage of the mass culture in vivo, however, exhibited increased tumorigenicity and v-Ha-ras expression. To examine and compare the molecular events involving the ras oncogene during cell transformation in vitro and subsequent tumor formation in vivo, clonal cell populations were isolated from the v-Ha-ras-transformed mass culture. While the clonal cell lines exhibited diverse tumorigenic profiles, these differences did not correlate with v-Ha-ras expression. However, the expression of the activated ras gene, while not necessary for growth in vitro, did appear to be associated with a selective growth advantage in vivo. In addition, the modulation of gene amplification ability in these cells was not associated with the induction of tumorigenicity or v-Ha-ras expression. ©1994 Wiley-Liss, Inc.     

Confluent BALB/c 3T3 cells monolayer provides selective and efficient growth of neoplastic cells

In an attempt to further characterize non-irradiated contact-inhibited confluent monolayer of BALB/c 3T3 cells (= Contact-Sensitive Plates; CSP) as substrata for in vitro drug sensitivity testing, we compared the efficiency of colony formation with panels of cell lines on CSP with that on plastic dishes or in agar. Tumorigenicity in athymic nude mice was also examined. We found that: (1) HeLa cells, 2 esophageal cancer lines, rat 3Y1 fibroblasts transformed by either adenovirus type 12, mouse polyoma virus, Rous avian sarcoma virus, or plasmid DNA carrying v-Ha-ras oncogene all formed colonies on CSP and in agar and at the same time was tumorigenic. The efficiency of colony formation on CSP proved always to be higher than that in agar. (2) None of the 4 "normal" fibroplastic cell lines formed colonies on CSP or in agar and were tumorigenic. (3) Simian virus 40 and adenovirus E1A gene transformed rat 3Y1 fibroblasts formed colonies on CSP but not in agar, and were not tumorigenic. Therefore, CSP was found to provide selective and efficient growth of neoplastic cells when compared to other substrata and is also helpful in detecting incompletely transformed cells.     

Hormone sensitivity in vitro and in vivo of v-ras-transfected MCF-7 cell derivatives

Human mammary carcinoma cell lines (MCF-7) were analysed for their hormone sensitivity before and after transfection with a v-Ha-ras oncogene or with a neomycin-resistance gene followed by selection in vitro or in vivo. Our aim was to test how the expression of the ras oncogene would influence the estradiol sensitivity of MCF-7 cells. In culture, MCF-7 cells expressing the viral p21 oncogene product, as compared to parental MCF-7 cells and their control derivatives, showed lower levels of a 67-kDa estrogen receptor. Progesterone receptors, however, remained sensitive to up-regulation by estrogens. The oncogene-expressing cells were less sensitive than all controls to stimulation of proliferation by 10(-8)M estradiol or to inhibition of proliferation by 2-CH3-4-OH tamoxifen, and this was not dependent upon the type of culture medium used. After s.c. or i.p. injection into female athymic nude mice, ovariectomized or left intact, the growth of MCF-7 cells expressing the ras oncogene product and of all control cells was sensitive to stimulation by estrogen supplementation. Conversely, cell lines derived from tumors generated with long latency in untreated athymic nude mice by v-ras-expressing MCF-7 cells showed efficient formation of quickly growing tumors in the absence of estrogen supplementation. No differences were observed in invasion and metastasis of the different MCF-7 cell types injected into athymic nude mice that were supplemented with estrogens or not.     

A mechanism of hyperthermia-induced apoptosis in ras-transformed lung cells

Lung cancer, the leading cause of cancer-related deaths in both men and women, is the consequence of disordered apoptosis, induction of which may have therapeutic utility. Hyperthermia has been identified as a stimulus for apoptosis. We investigated the mechanism of hyperthermia-induced cell death in ras-transformed lung cells. Effect of hyperthermia (43 degrees C for 180 min) was compared between two cell lines, an immortalized (sv-40) normal human bronchial epithelial (BEAS2-B) and its malignant transformed (H-ras transfected) counterpart (BZR-T33). Survival after hyperthermia: 7-d growth culture BEAS2-B, 1.03 +/- 0.007 and BZR-T33, 0.39 +/- 0.008 (P < 0.05); clonogenic assays BEAS2-B, 0.76 +/- 0.003 and BZR-T33, 0.41 +/- 0.004 (P < 0.05). Hoechst positive (apoptotic) cells: BEAS2-B, 11 +/- 3% and BZR-T33, 78 +/- 5% (P < 0.05). TUNEL, DNA fragmentation, and Annexin-V all corroborate this result. Western blot comparing the effect of hyperthermia in BZR-T33 cells to BEAS2-B cells revealed: TRAIL and FAS-L displayed significant increases (threefold and twofold, respectively); caspase-3 showed a decrease in uncleaved form and an increase in cleaved form, and a 50-fold increase in activity effectively blocked with the caspase-3 inhibitor DEVD-fmk; caspase-9 showed near depletion of uncleaved; poly (ADP-ribose) polymerase (PARP) degradation was clearly visible during heating. After hyperthermia, gene expression demonstrates a 5.7-fold increase in TRAIL and insignificant changes in tumor necrosis factor-alpha (TNF-alpha), FAS-L, and caspases 3, 8, 9 in transformed cells. Data demonstrated that hyperthermia induces apoptosis in transformed cells, and that apoptosis is mediated by caspase-3 as a result of activation of cell-death membrane receptors of the tumor-necrosis-factor family. In summary, these data suggest that hyperthermia could become an additional modality in the multidisciplinary approach to the treatment of lung cancer.     

Quantitative cell dispersion analysis: new test to measure tumor cell aggressiveness

Tumor progression requires the dispersion of epithelial cells from neoplastic clusters and cell invasion of adjacent stromal connective tissue. Aiming at demonstrating the precise relationships between cell dispersion and cell invasion, related respectively to expression of E-cadherin/catenin complex and matrix metalloproteinases (MMPs), we developed an original in vitro model of cell dispersion analysis. Our study reports the validation of this model that allowed us to analyze and quantify the cell cohesion level by means of time-lapse videomicroscopy and computer analysis based on the observation of spatial and temporal cell distribution. Our model was able to distinguish 2 groups among different human bronchial and mammary epithelial cells previously characterized for the expression of E-cadherin/catenin complex and MMPs and their invasive capacity in the Boyden chamber assay. The first group (16HBE14o(-), MCF-7, T47D) that expressed membranous E-cadherin and beta-catenin, and was negative for MMP-2 expression and non-invasive, displayed a highly cohesive pattern corresponding to a cluster spatial distribution. The second group (Beas2B, BZR, BZR-T33, MDA-MB-231, MDA-MB-435, BT549 and HS578T) that was invasive and showed lack of expression of E-cadherin and a cytoplasmic redistribution of beta-catenin, displayed a dispersed pattern corresponding to a random spatial distribution. Downregulation of E-cadherin by a blocking antibody induced a more random distribution. Conversely, expression of E-cadherin by cDNA transfection induced a cluster distribution. Moreover, tumor cell lines that co-expressed MT1-MMP and MMP-2 (Beas2B, BZR, BZR-T33, MDA-MB-435, BT549 and HS578T) showed a more dispersed pattern than tumor cell lines that did not express MMP-2 (MDA-MB-231). In conclusion, we demonstrated that the spatial group behavior of cell lines, i.e., their cohesion/dispersion ability, reflects their invasive properties. Thus, this model of cell dispersion analysis may represent a new test to measure tumor cell aggressiveness.     

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.     

Isolation and characterization of ras-transfected BALB/3T3 clone showing morphological transformation by 12-O-tetradecanoyl-phorbol-13-acetate

The transformation frequency of mouse BALB/3T3 cells was significantly enhanced after transfection with an activated ras oncogene (v-Ha-ras) followed by treatment with a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), suggesting that the ras oncogene acted as an initiator in two-stage carcinogenesis. A cell clone (Bhas42) containing the ras oncogene was isolated from the ras-transfected BALB/3T3 cells. Bhas42 cells were flat and showed contact inhibition, but the addition of TPA to quiescent Bhas42 cultures resulted in a dramatic change of cell morphology to spindle shape, doubling of the cell population, and increased DNA synthesis.     

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.     

Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.     

甲醛对人支气管上皮细胞系染色体不稳定性的研究

背景与目的： 为探讨染色体不稳定性与细胞恶性转化之间的关系,揭示化学致癌的机制,本研究以甲醛为诱导剂,研究化学致癌物甲醛作用于人支气管上皮细胞系后,对人支气管上皮细胞染色体稳定性的影响。 材料与方法： 用甲醛作为诱导剂,以液体染毒方式处理细胞,检测染毒后细胞的LC50,以低剂量（20% LC50）对细胞进行诱导并筛选诱导克隆。然后采用细胞遗传学方法（G带染色法）考察染毒后甲醛诱导的人支气管上皮细胞系（BEAS-2B）染色体畸变的情况。 结果： 甲醛作用后,诱导细胞的核型由2倍体转化为近2倍体、非整倍体和多倍体等核型同时存在。诱导细胞染色体稳定性降低,呈现大量染色体畸变,包括染色体丢失、内复制、易位、断裂、双/三着丝粒,同时伴有大量非稳定性畸变。 结论： 甲醛可影响BEAS-2B细胞染色体的稳定性,使其发生畸变并最终使细胞向恶性化方向发展。     

Oncogenic potential of erbB-2 in human mammary epithelial cells.

Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi-solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb-2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony-forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered.     

Identification of genes expressed differentially in an in vitro human lung carcinogenesis model

Lung cancer is the deadliest among all cancers in the US for both men and women. Early detection of premalignant lesions or tumors appears to be a promising approach to reducing the morbidity and mortality from lung cancer because the survival of early stage lung cancer patients is better than that of patients with advanced cancers. We approached the identification of early biomarkers of human lung carcinogenesis, by combining the use of cDNA array and an in vitro human lung carcinogenesis model that consists of normal (NHBE), immortalized (BEAS-2B and 1799), transformed (1198) and tumorigenic (1170-I) human bronchial epithelial (HBE) cells. We hypothesized that certain genes that are expressed differentially among these cells can serve as both biomarkers for early detection and targets for intervention. Nineteen genes were downregulated and six were upregulated in tumorigenic 1170-I HBE cells compared to normal HBE cells (NHBE) using cDNA array. Downregulated genes encode cell-to-cell and cell-to-matrix adhesion-related proteins, calcium-binding proteins and some enzymes or growth factor-related proteins. The functions of upregulated gene were more variable. Similar expression of selected genes was found in different nonsmall cell lung cancer cell lines analyzed by Northern blotting. Furthermore, the differential expression of some genes was also observed by in silico analysis of database of human lung tumors. The differentially expressed genes encode calcium-binding proteins and cell-cell and cell-matrix adhesion proteins. Furthermore, Northern blotting analysis of RNA from different cell lines comprising an in vitro carcinogenesis model including normal, immortalized, transformed, and tumorigenic cells indicated that the expression of subsets of the identified genes changed at different stages of carcinogenesis. These data show that the in vitro carcinogenesis cell system could be useful for discovering potential biomarkers of early and late stages of lung carcinogenesis and targets for chemoprevention.     

Cumulative gene and chromosome alterations associated with in-vitro neoplastic transformation of human cervical cells

The development of cancer is a multistep process requiring cumulative genetic alterations. An in vit ro model utilizing human cervical cells and papillomaviruses (HPV) that mimics human cervical cancer has been developed. Chromosome and gene alterations associated with distinctive stages of neoplastic transformation were demonstrated with an exocervical cell line obtained after sequential transfection with recombinant HPV-16 DNA and v-Ha-ras oncogene. Acquisition of immortality after HPV-16 transfection was associated with aneuploidy, structural changes of chromosomes 8, 10, 17, 19, 20, and 21, as well as proto-oncogene alterations. HPV-16 DNA was localized to two sites on chromosome 21, with one site at 21q22.2-22.3 near the ets-2 proto-oncogene. Ets-2 as well as c-myc gene mRNA levels were elevated in HPV immortal cells compared to primary nontransfected exocervical strains. Although the HPV-immortalized cells had several features characteristic of malignant cells, they lacked tumorigenic potential. Tumorigenicity occurred after transfection with v-Ha-ras oncogene, which was found stably integrated on chromosome 12 at the telomeric band q24.3. The tumorigenic line had additional clonal chromosomal abnormalities; consisting of multiple deletions involving regions of chromosomes 1p/q, 3p, 9q, loss of one copy of chromosome 11, and a complex rearrangement of chromosomes 8 and 13 as shown by in situ suppression hybridization with whole chromosome probes. Loss of tumor suppressor genes on deleted regions may have contributed to the acquisition of tumorigenicity. The genetic changes observed in these cells parallel those found in cervical carcinomas, demonstrating the validity of the in vitro model for studying the multistep progression resulting in cervical carcinoma.