Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway.

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.

Cycloheximide-dependent reversion of human cells transformed by MSV and chemical carcinogen.

The protein synthesis inhibitor cycloheximide, at a concentration of 0.08 microgram per milliliter, induced flat morphology within 24 to 48 hours and low saturation density in human osteosarcoma cells transformed by Kirsten murine sarcoma virus (Ki-MSV) or N-methyl-N' nitro-N-nitrosoguanidine. Removal of the protein synthesis inhibitor caused both transformed cells to revert to the transformed phenotype. The demonstration of cell-surface antigens, cross-reacted with antiserums induced by extracts of both types of transformed human cells, was dependent on the presence or absence of cycloheximide in the culture medium. The results show that protein synthesis is required to maintain the transformed state in virally or chemically transformed human cells.

Neoplastic conversion of human keratinocytes by adenovirus 12-SV40 virus and chemical carcinogens.

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12 and simian virus 40 (Ad 12-SV40) were transformed by treatment with the chemical carcinogens N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes.

Revertants of human cells transformed by murine sarcoma virus.

Revertants of nonproducer human osteosarcoma (NP/KHOS) cells induced by Kirsten murine sarcoma virus were isolated after incubating at high temperature (40.5 degrees C) overnight and subcloning at 36 degrees C. The morphologic variants, from which murine sarcoma virus could no longer be rescued, had growth properties similar to those of the nontransformed, parent human osteosarcoma cells and did not release RNA-dependent DNA polymerase activity. These revertants were nontumorigenic in nude mice. The revertants supported leukemia virus growth and showed an enhanced sensitivity to murine sarcoma virus superinfection. Thus, the revertants were from human cells transformed by an oncogenic RNA virus.

Neoplastic transformation of human epidermal keratinocytes by AD12-SV40 and Kirsten sarcoma viruses.

Recent investigations have begun to dissect the number and nature of genetic alterations associated with cancer cells. In the present study, primary human epidermal keratinocytes acquired indefinite life-span in culture but did not undergo malignant conversion in response to infection with a hybrid of adenovirus 12 and simian virus 40. Addition of Kirsten murine sarcoma virus, which contains a K-ras oncogene, to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary epithelial cells in culture and support a multiple-step process for neoplastic conversion.

Characterization of the androgen receptor in a benign prostate tissue-derived human prostate epithelial cell line: RC-165N/human telomerase reverse transcriptase.

The majority of prostate epithelial cell lines stably expressing wild-type (wt) or mutant (mt) androgen receptor (AR) are derived from metastatic prostate cancers. Therefore, the wt AR-expressing RC-165N/human telomerase reverse transcriptase (hTERT) cell line derived from the benign prostate tissue of an African-American patient provides a unique opportunity to assess the functional status of AR in a cellular context not studied before. Although androgen-induced expression of known androgen responsive genes such as PMEPA1, and NDRG1 was observed in RC-165N/hTERT, this cell line expresses prostate-specific antigen (PSA) at significantly lower levels. Chromatin immunoprecipitation assay revealed androgen-dependent binding of AR to androgen response elements of PSA, PMEPA1 and NDRG1 genes. Similarities, as well as differences were noted in the expression of androgen responsive genes between RC-165N/hTERT and LNCaP cells. Comprehensive evaluations of AR functions in RC-165N/hTERT cells suggest that whereas some features of known AR functions are maintained in this benign prostatic tissue-derived cell line, other AR functions are not retained. Objective evaluations of similar cell lines will lead to the understanding of AR functions in prostate growth and differentiation.

Inhibition of chemically induced carcinogenesis of canine cells in vitro by infection with mouse xenotropic type C virus.

C57L mouse xenotropic type C virus infection inhibited N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced in vitro transformation of embryo cells from inbred beagles. Treatment with MNNG induced in vitro neoplastic transformation in uninfected canine cells but not in C57L virus-infected canine cells; the C57L virus-infected canine cells not treated with MNNG also remained untransformed. In this dog cell system, preinfection with a C57L mouse xenotropic type C virus inhibited in vitro neoplastic transformation induced by MNNG.

Transformation of rat liver epithelial cells by Kirsten murine sarcoma virus.

We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.

Characterization of virus-free guinea pig tumors induced by Kirsten sarcoma virus.

Tumors were induced by Kirsten sarcoma virus (KiSV) in an inbred guinea pig, strain 13. The tumor cells were established in culture and characterized. The KiSV-induced sarcoma cells were virus-free and nonproducing; however, they contained resuable sarcoma genome. A type B guinea pig retravirus was readily activated from the tumor cells after induction with 5-bromodeoxyuridine (BUDR). BUDR induction of guinea pig retravirus was further enhanced by treatment with dexamethasone, a synthetic glucocorticoid hormone.

Survival of human cells in the aggregate form: potential index of in vitro cell transformation.

The ability of cell populations to survive in the aggregate form was compared to colony formation in soft agar and tumorigenicity in nude mice. Nontumorigenic human osteogenic sarcoma (HOS) cells, which formed colonies in soft agar, could not survive in the aggregate form. Tumorigenic HOS cell lines, which also formed colonies in soft agar, survived and proliferated in the aggregate form. Other cell types were tested with the same results. This approach, based on cell survival in the aggregate form, may provide an additional, reliable method for predicting the tumorigenic status of a cell population.

Transformation of human osteosarcoma cells by a chemical carcinogen.

A human osteosarcoma clonal cell line (TE-85, clone F-5) was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene or dimethyl sulfoxide (control). Cells treated only with the carcinogen underwent morphologic alteration in vitro, and one of these altered cell lines produced tumors subcutaneously and intracerebrally when injected into NIH nude mice.

Thyroid hormone modulation of transformation induced by Kirsten murine sarcoma virus.

We have investigated the effect of triiodothyronine (T3) on the transformation of normal rat kidney (NRK) cells by the Kirsten strain of murine sarcoma virus (Ki-MSV). When NRK cells were grown and infected with Ki-MSV in medium lacking T3, the yield of transformed foci was about one-half that observed in the cultures supplemented with T3. Individual foci appeared somewhat later in cells grown out in medium devoid of T3. The yield of Ki-MSV released from transformed NRK cells was lower when these cells were maintained in T3-depleted medium. The results cannot be attributed to cell growth modification by T3. Normal and Ki-MSV-transformed NRK cells grew equally well in mono-layer culture in medium containing or lacking T3. Selective maintenance and removal of T3 during various phases of the transformation process indicated that T3 exerted its maximum effect on transformation rates when added to the medium 24 h prior to virus infection. T3 was less effective in modulating transformation when added simultaneously with virus infection and was ineffective if added 24 h after virus infection. The results indicate that thyroid hormone is a required factor for optimal transformation by Ki-MSV and that the hormone exerts its effects during the early phase of Ki-MSV-induced transformation.

Malignant human papillomavirus type 16-transformed human keratinocytes exhibit altered expression of extracellular matrix glycoproteins.

We found that keratinocytes immortalized with human papillomavirus (HPV) type 16 DNA and malignantly converted by H-ras transfection (HPK-1A/ras) exhibit an enhanced ability to synthesize a fibronectin-containing extracellular matrix. Gene expression of fibronectin and thrombospondin was increased in tumorigenic keratinocytes compared to control and immortalized keratinocytes, 6- and 9-fold, respectively. Increased production of soluble and cell surface-associated fibronectin was not specific for HPV 16 transformed keratinocytes. Ad12-SV40-immortalized keratinocytes malignantly converted by H-ras transfection (RHEK-1/ras) also exhibited enhanced expression of fibronectin and thrombospondin, as well as pro-alpha 1 type I collagen. Steady state mRNA levels for autocrine growth-regulatory factors, transforming growth factors alpha and beta 1, were increased in Ad12-SV40 but not HPV 16-transformed human keratinocytes. We then determined whether increased production of fibronectin was associated with aberrant differentiation of transformed keratinocytes. Less than 10% of the HPV 16-transformed cells produced cornified envelopes after suspension-induced differentiation compared to 70% of normal keratinocytes. However, immortalization by HPV 16 DNA was sufficient to confer a differentiation-defective phenotype. Both involucrin mRNA and protein levels were decreased 8-fold in HPV 16-immortalized keratinocytes compared to normal cells and malignant conversion further attenuated involucrin levels. These studies demonstrate that aberrant differentiation is an early event in the transformation of the human keratinocytes and is not the result of enhanced expression of the extracellular matrix proteins. Unlike transformed fibroblastic cell types, up-regulation of fibronectin gene expression and matrix formation is a consistent characteristic of malignantly converted human keratinocytes.

Neoplastic transformation of immortalized human keratinocytes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most powerful carcinogen ever tested in animals. Recent epidemiological studies have suggested its carcinogenic potential in humans. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12-simian virus 40 (Ad12-SV40) were transformed by exposures of TCDD equal to or greater than 0.1 nM for 2 wk. These transformed cells showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas no such transformation phenotypes were observed with exposures of less than 0.1 nM for 2 wk. Primary human epithelial keratinocytes exposed to various concentrations of TCDD failed to show any evidence of transformation. Induction of aryl hydrocarbon hydroxylase activity was dose dependent, as was transformation. Thus, the carcinogenicity of TCDD in this human cell system appears to be an Ah receptor-mediated process. The present study represents the first evidence of neoplastic conversion of human cells exposed to this environmentally important chemical.

Activated H-ras oncogenes in human kidney tumors.

Two H-ras oncogenes were detected by NIH/3T3 transfection assay out of 16 primary kidney tumors, 15 renal cell carcinomas (RCC), and one transitional cell carcinoma in 16 patients. Analysis of ras Mr 21,000 protein suggested single point mutations within codon 12 and 61 in each case. The restriction endonuclease analysis of H-ras gene at codon 12 confirmed this in one of them, and the remaining 15 tumors did not have a mutation at this site. DNAs from the noncancerous portions of the kidney with codon 12 mutated tumor, but not leukocytes from the same patient, showed an abnormal resistance to the endonucleases MspI and HpaII, suggesting a presence of codon 12 mutated H-ras gene in the noncancerous cells. No amplification of ras genes was detected in the 16 tumors analyzed. In one of eight tumors from patients heterozygous for H-ras related BamHI restriction fragments, one allele was lost in the tumor but not in the noncancerous portion of the same kidney. Although cytogenetic studies have previously suggested nonrandom involvement of c-raf-1 gene in RCC, no abnormality in the size nor amount of raf transcript was detected in the 15 RCCs. Our results thus indicated that the genetic lesions affecting ras genes do occur in human RCC, and probably serve as one of multisteps in the carcinogenic process.

A novel human cell culture model for the study of familial prostate cancer.

Research into molecular and genetic mechanisms underlying familial prostate cancer would be greatly advanced by in vitro models of prostate tumor cells representing primary tumors. We have successfully established an immortalized human prostate epithelial cell culture derived from primary tumors of familial prostate cancer patients with telomerase. The actively proliferating early-passaged 957E cells were transduced through infection with a retrovirus expressing the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT). A high level of telomerase activity was detected in 957E/hTERT cells, but not in 957E cells. 957E/hTERT cells are currently growing well at passage 40, whereas 957E cells senesced at passage 5. 957E/hTERT cells exhibit epithelial morphology. Expression of an androgen-regulated prostate specific homeobox gene NKX3.1 and an epithelial cell-specific cytokeratin 8, but not prostate specific antigen or androgen receptor, was detected in 957E/hTERT cells. Prostatic stem cell antigen and p16 were also expressed in this line. 957E/hTERT cells showed growth inhibition when exposed to retinoic acid and transforming growth factor beta1, potent inhibitors of prostate epithelial cell growth. Chromosome analysis showed that the 957E/hTERT cell line (passage 10) was near diploid human male (XY), with most chromosome counts in the 44-46 range. However, there was random loss of chromosomes 8, 13, X, Y, and alteration in chromosome 4q. The late passage 957E/hTERT cell line (passage 32) was karyologically similar to the early passage 957E/hTERT cell line (passage 10) and also had the same alteration of 4q observed in the early passage 957E/hTERT cell line (passage 10) as well as a trisomy of chromosome 20. The well-characterized human cancer lines derived from such patients will be useful for the identification and characterization of prostate cancer susceptibility genes. This is the first documented case of an established human prostate cancer cell line from primary tumor of a familial prostate cancer patient.

Enhanced G2 chromatid radiosensitivity, an early stage in the neoplastic transformation of human epidermal keratinocytes in culture.

A deficiency in DNA repair, manifest as enhanced chromatid radiosensitivity during the G2 phase of the cell cycle, together with a proliferative stimulus such as that provided by active oncogenes may be necessary and sufficient for the malignant neoplastic transformation of human keratinocytes in culture. Normal epidermal keratinocytes established as continuous cell lines by transfection with pSV3-neo or infection with adeno 12-SV40 hybrid virus developed enhanced G2 chromatid radiosensitivity after 18 passages in culture. In contrast to cells from primary or secondary culture, these cells could be transformed to malignant neoplastic cells by infection with Kirsten murine sarcoma virus containing the Ki-ras oncogene or in one line by the chemical carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine; both of these agents produced a marked proliferative response. Cytological heterogeneity and karyotypic instability characterized the cells during their progression to neoplasia. These results are interpreted in terms of a mechanism for neoplastic transformation.

Reversal of hypercalcemia with the vitamin D analogue EB1089 in a human model of squamous cancer.

EB1089, an analogue of 1,25 dihydroxyvitamin D with low calcemic activity is a potent inhibitor of parathyroid hormone-related peptide (PTHRP) production in vitro. The purpose of the present study was to determine whether EB1089 could reverse established hypercalcemia in BALB C nude mice implanted s.c. with a human epithelial cancer previously shown to produce high levels of PTHRP in vitro. Total plasma calcium was monitored before and after tumor development and increased steadily when the tumor reached > or =0.5 cm3. When total calcium was 22.85 mmol/liter, animals were treated with a constant infusion of EB1089 or vehicle alone for a period of 2 weeks. A significant and sustained reduction of plasma calcium from 3.2+/-0.1 to 2.7+/-0.08 (P < 0.01) mmol/liter was observed during infusion with EB1089. In contrast, calcium levels in vehicle-treated animals continued to rise during the infusion period. Tumor growth velocity also slowed significantly after the administration of EB1089 as compared with vehicle-treated animals. Plasma PTHRP levels measured at the end of the 2 weeks' infusion period were significantly lower in animals treated with EB1089 as compared with animals treated with vehicle alone (44+/-8 pg/ml versus 194+/-35 pg/ml, P < 0.001). These results, therefore, demonstrate that EB1089 can reverse established hypercalcemia in a human model of squamous cancer.

Evidence for the multistep nature of in vitro human epithelial cell carcinogenesis.

In keeping with the multistep development of human cancer in vivo, a stepwise approach to neoplastic transformation in vitro presents a reasonable strategy. We have recently developed an in vitro multistep model suitable for the study of human epithelial cell carcinogenesis. Upon infection with the adenovirus 12-simian virus 40 hybrid virus, primary human epidermal keratinocytes acquired an indefinite life span in culture but did not undergo malignant conversion. Subsequent addition of Kirsten murine sarcoma virus and human ras oncogene or chemical carcinogens (N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline 1-oxide) to these cells induced morphological alterations and the acquisition of neoplastic properties. Subsequently it was found that this line could be transformed neoplastically by a variety of retrovirus-containing H-ras, bas, fes, fms, erbB, and src oncogenes. In addition, we found that the immortalized human epidermal keratinocyte (RHEK-1) line can be transformed neoplastically by exposure to ionizing radiation. Thus, this in vitro system may be useful in studying the interaction of a variety of carcinogenic agents and human epithelial cells. These findings demonstrate the malignant transformation of human primary epithelial cells in culture by the combined action of viruses, oncogenes, chemical carcinogens, or X-ray irradiation and support a multistep process for neoplastic conversion.

Amplification, overexpression, and rearrangement of the erbB-2 protooncogene in primary human stomach carcinomas.

Four of 51 primary gastric carcinomas exhibited amplification of the erbB-2 protooncogene ranging from 2- to 8-fold. In three cases gene amplification affected erbB-2 alleles of normal gene structure as determined by Southern blot analysis. In addition, one tumor displayed gene amplification of an apparently rearranged erbB-2 allele. Analysis of the rearranged allele revealed a structural alteration consistent with an internal deletion of approximately 2 kilobase pairs within the erbB-2 gene. Quantitation of erbB-2 mRNA in these tumors demonstrated that gene amplification coincided with overexpression of erbB-2 mRNA ranging from 8- to 32-fold above levels observed in stomach tumors without gene amplification. Furthermore, in one of the tumors with amplified normal size erbB-2 restriction fragments, elevated erbB-2 mRNA levels consisted of the normal size 5-kilobase transcript. Thus, overexpression of normal size erbB-2 mRNA accompanies gene amplification in primary stomach tumors. In addition, evidence for erbB-2 gene rearrangement suggests a role for such alteration in the development of certain gastric carcinomas.