Loss of transforming growth factor beta 1 (TGF-beta 1)-induced growth arrest and p34cdc2 regulation in ras-transfected epithelial cells.

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of mink lung epithelial (CCL64) cell growth in culture. The fact that many transformed epithelial cells have escaped from negative growth control by TGF-beta 1 suggests that transfected epithelial cells may be an appropriate model for investigating the growth-inhibitory mechanism of TGF-beta 1. We transfected CCL64 cells with a mouse c-myc oncogene (pSVc-myc1), a mutated Harvey-ras (Ha-ras) oncogene, or a combination of both. The results indicate that cells transfected with c-myc alone exhibit normal morphology and maintain sensitivity to TGF-beta 1 growth arrest, but are unable to form colonies in soft agar in the presence or absence of TGF-beta 1. Cells transfected with Ha-ras, or co-transfected with c-myc, display a transformed morphology, grow spontaneously under anchorage-independent conditions and acquire a complete resistance to growth inhibition by TGF-beta 1. Affinity cross-linking of [125I]TGF-beta 1 to cell-surface receptors from these transfectants revealed that all three TGF-beta receptor types were present and no significant differences in [125I]TGF-beta 1 labeling of these receptors was observed. Since we have previously demonstrated that modulation of p34cdc2 kinase is a marker for TGF-beta 1 growth inhibition, we investigated p34cdc2 activity in the CCL64 transfected clones. The results show that in the control CCL64 cells and in the myc-transfected clones TGF-beta 1 regulation of p34cdc2 activity is maintained. In the ras- and ras + myc-transfected cells p34cdc2 phosphorylation and histone H1 kinase activity is significantly increased and regulation by TGF-beta 1 is lost.     

Isolation and characterization of Kirsten murine sarcoma virus-transformed mouse keratinocytes resistant to transforming growth factor beta

BALB/MK (MK) is a continuous murine keratinocyte line whose cells are strictly dependent on exogenous epidermal growth factor (EGF) for growth in culture. A derivative cell, KC, resulted from Kirsten murine sarcoma virus transformation, and these cells no longer require EGF for their growth. Despite differences in MK and KC growth conditions, both cell lines are growth inhibited by picomolar concentrations of transforming growth factor-beta (TGF-beta). When MK and KC cells were maintained in the presence of TGF-beta, resistant variants eventually proliferated only from the KC population. In an attempt to determine the mechanism of development of TGF-beta resistance, the TGF-beta-resistant cells (KCR cells) were compared with TGF-beta-sensitive KC cells with regard to growth properties, TGF-beta 1 binding characteristics, and gene expression. KCR cells continued to synthesize DNA and proliferated in the presence of TGF-beta 1 concentrations up to 2 nM, which was 500-fold greater than the ED50 for the sensitive cells. Although the KCR cells possess similar receptor numbers and affinity for TGF-beta 1, we observed differences in affinity cross-linking studies. The KCR cells expressed more of the type III, high molecular weight cell surface binding protein and less of the type II than the KC cells. The type I moiety was clearly altered to a smaller size in some, but not all, KCR cells. In gene regulation studies, there was no apparent difference in c-Ki-ras and v-Ki-ras mRNA levels in the KC and KCR cells. Additionally, expression of TGF-alpha and TGF-beta 1 mRNA was similar in MK, KC, and KCR cells. The expression of proliferation-associated genes, such as c-myc and MGSA/c-gro/kc, which were markedly decreased by TGF-beta 1 in the MK and KC cells, was not altered by TGF-beta 1 in the KCR cells. The data suggest that the loss of TGF-beta 1 responsiveness in the KCR cells was due to an alteration in the TGF-beta receptor that did not permit signal transduction, although the existence of postreceptor alterations cannot be excluded.     

Mid-G1 arrest and epidermal growth factor independence of ras-transfected mouse cells

Transfection of C3H/10T1/2 cells with either a c-myc or an activated c-Ha-ras gene decreased the cellular dependence for serum-derived factors to proliferate in monolayer. The c-myc-transfected cells did, however, require a high plasma concentration for significant growth, while the ras transfectants grew extremely well in either low or high concentrations of either plasma or serum. Stimulation of quiescent cultures with purified growth factors demonstrated that c-myc transfection did not alter qualitatively or quantitatively the requirement for both epidermal growth factor (EGF) and insulin to progress to DNA synthesis. Cells transfected with either a ras gene alone or a combination of ras plus c-myc lost their dependence on EGF for DNA synthesis; cultures became committed to S phase in serum-free medium supplemented with insulin alone. The ras transfectants arrested in mid-G1, 6 h prior to S phase. The EGF independence of the ras transfectants is consistent with the mid-G1 arrest of these cells at a point(s) distal to the primary action of EGF in early G0-G1.     

Expression of TGF-alpha/EGF and TGF-beta receptors in human colon carcinoma cell lines

Previous studies have established that colon carcinoma cells secrete several polypeptide growth factors, including TGF-alpha/EGF and TGF-beta, suggesting that these and related molecules function in an autocrine/paracrine fashion to modulate tumor-cell growth. To investigate this possibility, we have studied the expression of transforming growth factor receptors in a panel of human colon carcinoma cell lines and in several untransformed epithelial cell populations. The results have revealed that neoplastic colon cells express receptors for both TGF-alpha/EGF and TGF-beta. Immunoprecipitation identified the TGF-alpha/EGF receptor as a structurally intact 170-kDa protein. No evidence for over-expression was found. TGF-alpha (and EGF) enhanced receptor autophosphorylation, indicating that these receptors were biochemically functional. TGF-beta blocked DNA synthesis in non-neoplastic epithelial cells but not in tumorigenic colon populations. There was no correlation with TGF-beta receptor number or dissociation constant. However, chemical cross-linking studies revealed a TGF-beta receptor subtype of 75 kDa in 3 of the 4 colon carcinoma cells which was undetectable in normal IEC epithelial cultures, suggesting a possible association between 75-kDa receptor expression and refractoriness to growth inhibition of TGF-beta. Together, these data support the concept that locally-produced growth regulators can function in an autocrine or paracrine manner to influence the proliferation of colon carcinoma cells.     

Progressive abrogation of TGF-beta 1 and EGF growth control is associated with tumour progression in ras-transfected human keratinocytes.

This study examined the response of human keratinocytes in different stages of transformation to exogenous TGF-beta 1 and EGF as well as their receptor and growth-factor expression. Cells of the spontaneously immortalized HaCaT cell line and c-Ha-ras transfected clones (I-6, I-7, II-3, II-4) exhibited different tumorigenic potentials when transplanted to athymic mice. HaCaT- and I-6 cells were non-tumorigenic, I-7 cells formed persisting epidermal cysts (benign tumours) and II-3 and II-4 cells developed into invasive squamous-cell carcinomas. TGF-beta 1 inhibited thymidine uptake in a dose-dependent manner, a progressive decrease in response being associated with an increasing malignant potential (HaCaT greater than I-6 greater than I-7 = II-4). HaCaT-cells and ras-clones expressed TGF-beta 1 mRNA at similar levels, but cells of increasing malignant potential secreted markedly less receptor-binding TGF-beta (HaCaT greater than I-6 = I-7 greater than II-3 greater than II-4) into the culture medium. Whilst ras-transfected cells expressed fewer TGF-beta receptors than HaCaT cells, there was little difference between TGF-beta receptor number or affinity between the 4 transfected cell clones. The same was true for the TGF-beta receptor types, but Type-II receptors were expressed at lower levels by the malignant clones II-3 and II-4. When HaCaT and ras-transfected cells were investigated for their response to exogenous EGF, cells were refractory (I-7, II-4), partially stimulated (I-6) or fully stimulated (HaCaT). Cells with increasing malignant potential produced increasing amounts of endogenous TGF-alpha (II-4 = II-3 greater than I-7 = I-6 greater than HaCaT). All tumorigenic ras clones expressed higher mRNA levels than HaCaT-cells. Ras-transfected clones expressed fewer high- and low-affinity EGF receptors than HaCaT cells with a tendency toward increased numbers of high-affinity EGF receptors associated with increasing malignant potential (II-4 = II-3 greater than I-7 greater than I-6) but these changes were associated with a progressive decrease in receptor affinity. The results indicate that tumour progression in human epidermal keratinocytes transfected with c-Ha-ras is associated with a progressive abrogation of TGF-beta 1 and EGF growth control. They suggest that the increased autonomous growth potential associated with advanced stages of epithelial tumour progression can be defined more closely using a cellular profile of TGF-beta and EGF.     

Overexpression of H-ras oncogene induces resistance to the growth-inhibitory action of transforming growth factor beta-1 (TGF-beta 1) and alters the number and type of TGF-beta 1 receptors in rat intestinal epithelial cell clones.

In this report, we utilize rat intestinal cell (IEC-18) clones expressing an activated human H-ras gene to investigate the relationship between malignant transformation and growth control by transforming growth factor beta (TGF-beta). We demonstrate that clones expressing high levels of H-ras oncogene lose sensitivity to the growth inhibitory action of TGF-beta. The loss of sensitivity is related to the degree of H-ras expression and is shown to be a direct consequence of H-ras expression through the use of a clonal cell line with inducible expression of activated H-ras. Co-incident with the loss of growth inhibition, ras-expressing clones display an altered expression of TGF-beta-binding proteins as detectable by [125I]TGF-beta cross-linking. While IEC-18 cells express type II (92 kDa) binding protein predominantly, H-ras expression induces a shift to predominantly type I (69 kDa) binding protein expression.     

Transforming growth factor-beta and response to anticancer therapies in human liver and gastric tumors in vitro and in vivo

Liver cancer and gastric cancer are the most common solid tumors worldwide. Transforming growth factor-beta (TGF-beta) production and lack of response to TGF-beta growth inhibitory effects have been associated with tumor progression and therapeutic resistance. HepG2, Hep3B, and SK-HEP-1 human liver cancer lines produce 3, 5.7, and 2.5 ng TGF-beta1; 1.4, 2, and 4 ng TGF-beta2 and 0.15, 0.2 and 0.22 ng TGF-beta3 per 107 cells (24 h). Expression of the TGF-beta type I receptor is 20x, 1x, and 0.6x the level in mink lung MvLu1 cells in the HepG2, Hep3B, and SK-HEP-1 cells, respectively. HepG2 and Hep3B cells do not express the TGF-beta type II receptor while SK-HEP-1 cells express 7x the level found in mink lung MvLu1 cells. Hs 746T, KATO III, RF-1, and RF-48 human gastric cancer cell lines produce 12. 5, 0.35, 0.4, and 0.4 ng TGF-beta1; 2.6, 0.95, 0.5, and 0.52 ng TGF-beta2 and 0.42, 0.17, 0.12, and 0.14 ng TGF-beta3 per 107 cells (24 h). Expression of TGF-beta type I receptor is 0.7x, 0.7x, 0.8x, 0.6x the level in mink lung MvLu1 cells in the Hs 746T, KATO III, RF-1 and RF-48 cells, respectively. KATO III cells are lacking in the TGF-beta type II receptor while Hs 746T, RF-1 and RF-48 cells express 10x, 0.8x, and 1x the levels in mink lung MvLu1 cells. The IC50 for TGF-beta1 is >10 ng/ml in all of these lines except RF-48 where TGF-beta1 is mitogenic. The response of the cell lines to radiation, doxorubicin, mitomycin C, cisplatin, 5-fluorouracil, methotrexate, and gemcitabine showed that SK-HEP-1 was the most drug resistant liver cancer cell line and KATO III was the most drug resistant gastric cancer cell line. Overall, there was no correlation between TGF-beta secretion in cell culture and sensitivity of the cells to anticancer agents. Increased TGF-beta1 levels were detectable in the plasma of nude mice bearing Hep3B and Hs 746T xenografts. Those tumors which secreted greater amounts of TGF-beta were more therapeutically resistant in vivo.     

Differential-effects of transforming growth-factor-Beta-1 on protein-levels of p21 waf and cdk-2 and on cdk-2 kinase-activity in human rd and ccl64 mink lung-cells

Currently, the cyclin-dependent kinase inhibitor p21 WAF-1 is considered to be a crucial downstream effector in the p53-specific pathway of negative growth control in mammalian cells. Wild-type p53, but not mutant forms of this protein, transactivate the WAF-1 gene. We show a correlation between growth-inhibition and induction of WAF-1 protein expression following transforming growth factor-beta 1 (TGF-beta 1) treatment of two human tumour cell lines devoid of wild-type p53 protein and in SV40-transformed WI38 fibroblasts. Inversely, TGF-beta 1 treatment of normal WI38 fibroblasts stimulates their growth and represses WAF-1 protein synthesis. As the mink lung epithelial CCL64 cell line is frequently used in TGF-B studies we included it in this study: TGF-beta 1 growth-inhibition is accompanied by induction of WAF-1 synthesis concomitantly with a reduction of cdk2 synthesis and of its histone kinase activity. However in the human tumour line RD, TGF-beta 1 did not affect cdk-2 protein levels but did reduce its histone kinase activity.     

Multiple effects on drug-sensitivity, genome stability and malignant potential by combinations of h-ras, C-myc and mutant p53 gene overexpression

Activation of specific oncogenes and inactivation of tumor suppressor genes play major roles in mechanisms leading to neoplastic transformation. The potential involvement of these genes in determining genome stability is an important issue. To examine the relationships between altered oncogene expression and the effects on genome stability, we have investigated the drug sensitivity properties of mouse 10T1/2 fibroblasts transfected with combinations of H-ras, c-myc and the proline 193 mutant form of p53. The relative colony forming efficiencies of these cells were investigated in the absence or presence of various concentrations of the chemotherapeutic agents, methotrexate, N-(phosphonacetyl)-L-aspartate (PALA) or hydroxyurea. The effects of altered oncogene expression were found to be drug and locus specific, and to lead to increased drug resistance (e.g. H-ras transfectants were significantly resistant to methotrexate or PALA), decreased drug resistance (e.g. H-ras/-myc transfectants were significantly less resistant to PALA or hydroxyurea than H-ras transfected cells), or to no significant change in drug sensitivity (e.g. H-ras transfected cells were not significantly different in sensitivity to hydroxyurea than non-transfected cells). Gene amplification was an important but not the only mechanism for drug resistance. Cells that were transfected with p53 (H-ras/p53 or H-ras/c-myc/p53) exhibited the greatest drug resistance properties with all three chemotherapeutic agents, in keeping with the important role of p53 in DNA repair and DNA amplification mechanisms. Although both H-ras/p53 and H-ras/c-myc/p53 groups exhibited very similar genome stability characteristics as determined by drug sensitivity results, they were significantly different in their abilities to produce transformed foci in vitro and lung metastases in vivo. The H-ras/c-myc/p53 transfected cells formed significantly higher numbers of transformed foci and exhibited a greater malignant potential. These results are consistent with observations that H-ras expression directly correlates with malignant potential, and that H-ras/c-myc/p53 transfected cells have higher H-ras expression than H-ras/p53 transfected cells. Alterations in genomic integrity through changes in onocogene expression play important roles in mechanisms determining drug sensitivity; in addition to genome destabilization, other events are critically involved in regulating transformed and malignant characteristics.     

Modulation of c-myc expression by transforming growth factor beta 1 in human hepatoma cell lines

The effects of transforming growth factor beta 1 (TGF-beta 1) on cell proliferation of human hepatoma cell lines, PLC/PRF/5 and Mahlavu, were investigated under serum-free conditions. DNA synthesis was strongly inhibited in the PLC/PRF/5 cells by addition of TGF-beta 1 (0.5 to 4.0 ng/ml), but remained unchanged in the Mahlavu cells. Also the expression of c-myc mRNA was suppressed by the addition of TGF-beta 1 in the PLC/PRF/5 cells but not in the Mahlavu cells. These results indicate that TGF-beta 1 might regulate cell growth, in part, by modulating c-myc expression, although there is no direct proof that c-myc expression is really relevant to DNA synthesis mediated by TGF-beta 1.     

Inhibition of topoisomerase IIalpha expression by transforming growth factor-beta1 is abrogated by the papillomavirus E7 protein

Transforming growth factor-beta (TGF-beta) protects normal cells from etoposide-induced cell death, yet the mechanism has remained speculative. Studies have shown that etoposide modifies the activity of the topoisomerase IIalpha (topo IIalpha) enzyme, thereby causing DNA damage and inducing cell death. Expression of topo IIalpha is necessary for etoposide-induced cell death, and peak expression of topo IIalpha normally occurs during the G2 phase of the cell cycle. We predicted that by arresting growth in the G1 phase, TGF-beta1 would prevent the induction of topo IIalpha expression that normally occurs subsequent to the G1-S transition, thereby protecting cells from etoposide-induced cell death. Accordingly, we hypothesized that the inhibition of topo IIalpha expression by TGF-beta1 would be dependent on the ability of TGF-beta1 to arrest cell cycle progression in G1. Using mink lung epithelial cells (MvlLu), we found that TGF-beta1 decreases topo IIalpha mRNA expression, and the decrease occurs as cells begin to accumulate in the G1 phase of the cell cycle. Topo IIalpha protein expression decreases subsequent to the fall in mRNA expression. In contrast, topo IIalpha expression is not affected by TGF-beta1 in cells that fail to undergo G1 arrest because of inactivation of the retinoblastoma tumor suppressor protein (pRb) by the papillomavirus type 16 E7 protein. Our studies suggest that inhibition of topo IIalpha by TGF-beta1 is the principal mechanism that protects mink lung epithelial cells (Mv1Lu) from etoposide-induced toxicity. Furthermore, the inhibition of topo IIalpha protein expression by TGF-beta1 is dependent on pRb-mediated cell cycle arrest, suggesting that TGF-beta1 will not reduce the sensitivity of pRb-deficient cancers to etoposide.     

Myc protein is differentially sensitive to quinidine in tumor versus immortalized breast epithelial cell lines

Quinidine regulates growth and differentiation in human breast tumor cells, but the immortalized mammary epithelial MCF-10A cell line is insensitive to quinidine. We found that a morphologically similar differentiation response was evoked by quinidine and c-myc antisense oligonucleotides in MCF-7 cells and this prompted us to investigate the actions of quinidine on c-myc gene expression. Myc protein levels were suppressed in human breast tumor cell lines, but not in MCF-10A cells, an observation that supports the hypothesis that suppression of c-myc gene expression is involved in the preferential growth and differentiation response of breast tumor cells to quinidine. Quinidine reduced c-myc mRNA levels in MCF-7 cells. Acute induction of c-myc mRNA by estradiol, as well as the c-myc response to sub-cultivation in fresh serum and H-ras driven elevations in c-myc mRNA were depressed by 50-60% in the presence of quinidine. Quinidine decreased c-myc promoter activity in MCF-7 cells in a transient reporter gene assay and a 168 bp region of human c-myc promoter (-100 to +68 with respect to the P1 promoter) was sufficient to confer responsiveness to quinidine. Quinidine is a potential lead compound for developing pharmacological agents to regulate Myc. In addition, the study of quinidine-regulated events is a promising approach to unravel differentiation control pathways that become disrupted in breast cancer.     

Isolation and characterization of a rat liver epithelial cell line resistant to the antiproliferative effects of transforming growth factor beta (type 1)

Rat liver epithelial cells resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF-beta 1) were isolated after 3 h exposure to 1.5 micrograms/ml of N-methyl-N'-nitro-N-nitrosoguanidine followed by continuous treatment with 1 ng/ml TGF-beta 1 for 6 weeks. In comparison to the parental or N-methyl-N'-nitro-N-nitrosoguanidine-exposed rat liver epithelial cells (concentration causing 50% inhibition of the rate of DNA synthesis, 0.25 ng/ml), these cells were 10-fold more resistant to the antiproliferative effects of TGF-beta 1 and exhibited resistance to growth inhibition by a highly purified liver-derived growth inhibitor, recombinant human tumor necrosis factor, and transforming growth factor beta 2. Single cell cloning of these resistant cells led to the isolation of a nontransformed clonal cell population (clone 11) which maintained stable resistance in the absence of TGF-beta 1 treatment. Binding of 125I-labeled TGF-beta 1 to rat liver epithelial cells and clone 11 cells was similar. Clone 11 cells exhibited a 5-10-fold resistance to the cytotoxins Adriamycin and vinblastine as assessed by a clonogenic assay. This drug resistance was accompanied by an increase in the steady state levels of the mRNAs for multidrug resistance gene (MDR-1), glutathione S-transferase-P, TGF-beta 1, and c-myc genes. The data presented here suggest an association between resistance to the growth-inhibitory effects of TGF-beta 1- and MDR-1-mediated multidrug resistance.     

Neoplastic transformation of human keratinocytes by polybrene-induced DNA-mediated transfer of an activated oncogene

Polybrene, in conjunction with dimethyl sulfoxide (DMSO) shock has been shown to increase the frequency of DNA-mediated gene transfer to mammalian cells as compared with the frequency obtained with calcium phosphate transfection. We have successfully adapted this procedure for use with human epidermal keratinocytes. Non-tumorigenic human epidermal epithelial cells immortalized by SV40 tumor antigen were neoplastically transfected, using Polybrene at a concentration of 10 micrograms ml-1, followed by a 4 min shock, with 30% DMSO, with a plasmid carrying the activated H-ras gene from the EJ bladder carcinoma cell line. The transfected cells showed morphological alterations and induced carcinomas when transplanted into nude mice. They contained integrated copies of the transfected H-ras gene and expressed high levels of the p21 protein. Polybrene-induced DNA transfection, therefore, offers the opportunity to transfer genes effectively into human epidermal keratinocytes and should accelerate the study of the interaction between oncogenes and human epithelial cells. This study appears to represent the first neoplastic conversion of nontumorigenic, immortalized human epidermal keratinocytes by an activated human oncogene.     

Loss of growth control by TGF-beta occurs at a late stage of mouse skin carcinogenesis and is independent of ras gene activation

The relationship between the expression of a mutant ras gene in epithelial cells and loss of responsiveness to the negative effects of transforming growth factor beta (TGF-beta) is presently unclear. We have investigated this question using a series of cell lines derived from benign and malignant mouse skin tumours which express mutant forms of the H-ras gene. Immortalised, non-tumorigenic mouse epidermal cells respond to TGF-beta by cessation of growth, whereas in a series of malignant carcinoma lines the response was substantially reduced. Introduction of a mutant H-ras gene into the immortalised cells did not lead to any appreciable change in TGF-beta responsiveness, suggesting that initiation of carcinogenesis by ras mutation does not directly alter growth control by this pathway. Of two non-tumorigenic papilloma lines tested which had mutant H-ras genes, one retained complete sensitivity to TGF-beta, whereas the other showed a similar response to carcinomas. We conclude that growth control by TGF-beta is lost at a relatively late stage of carcinogenesis in this system, and is independent of ras gene activation.     

Timing of induction of tgf-alpha in ras induced human bladder neoplastic progression

Introduction of a H-ras oncogene into an SV-40 immortalized human urothelial cell lines (SV-HUC) results in morphologically altered cell clones which acquire tumorigenic potential following serial passaging in culture. Early and late passage cells, from individual ras transfected clones exhibiting different tumorigenic potential, display increased growth factor synthesis in mitogenic assays. Northern blot analysis revealed induction of TGF-alpha mRNA concomitant with the introduction of a H-ras oncogene with no modulation in EGF receptor expression observed throughout neoplastic progression. Consistent with completion of an autocrine loop, down modulation and activation of EGF receptors was observed in early passage cells coincident with TGF-alpha expression. In this human urothelial progression model TGF-alpha secretion follows the introduction of a H-ras oncogene prior to the acquisition of tumorigenic potential.     

Tumour progression in experimental oral carcinogenesis is associated with changes in EGF and TGF-beta receptor expression and altered responses to these growth factors

This study examined the characteristics of premalignant oral epithelial cell lines derived from non-invasive palatal and lingual mucosa of rats painted with the carcinogen 4-nitroquinoline N-oxide (4NQO) in vivo. In contrast to normal keratinocytes, premalignant epithelial cells had an extended life span, were independent of 3T3 fibroblast support, and expressed variable anchorage independence in gel culture and tumorigenicity in athymic mice. The expression of these functional phenotypes did not correlate with the duration of 4NQO treatment. Keratinocytes from 4NQO-treated tissues predominantly had fewer epidermal growth factor (EGF) receptors than normal controls. The expression of high-affinity EGF receptors paralleled the emergence of the anchorage-independent phenotype and was markedly elevated in tumorigenic cell lines. Cell lines with an extended life span expressed fewer transforming growth factor beta 1 (TGF-beta) receptors than their normal counterparts though the loss of these receptors appeared to be unrelated to either anchorage independence or tumorigenicity. Normal keratinocytes were stimulated and inhibited, in a dose-dependent manner, by EGF and TGF-beta respectively. By contrast, a cell line that was immortal, anchorage dependent and non-tumorigenic showed reduced sensitivity to stimulation by EGF and was inhibited only by high concentrations of TGF-beta. Cells that were immortal, anchorage independent and tumorigenic, however, were refractory to EGF and were inhibited only by high concentrations of TGF-beta. There was no correlation between the expression of EGF or TGF-beta cell surface receptors and the response to ligand binding. The results show that tumour progression in rat oral epithelial cells is associated with a progressive independence of growth factor control. The number and distribution of EGF and TGF-beta receptors may be useful markers in more closely defining the stages of epithelial tumour progression.     

No acquisition of metastatic capacity of R1H rhabdomyosarcoma upon transfection with c-Ha-ras oncogene

The effect of the activated c-Ha-ras oncogene on invasiveness and formation of spontaneous metastases was studied using the rhabdomyosarcoma R1H of the rat. R1H tumor cells which are able to grow in vitro and produce tumors upon subcutaneous injection in syngeneic WAG/Rij rats were transfected with the c-Ha-ras (EJ) oncogene and the neomycin gene for selection. Two R1H cell lines harboring and expressing the human c-Ha-ras oncogene, one cell line containing the neomycin gene only, and the parent R1H cell line were compared. The expression of the transfected c-Ha-ras oncogene was assessed by Northern blot analysis and by flow cytometry using antibodies against ras p21. No difference in tumor growth rate and morphology was observed for the transfected and untransfected cell lines. Tumor volume doubling time was about 2 days in R1H-ras as well as in R1H parent tumors. Formation of spontaneous metastases was tested by excising the tumors when they had reached a volume of 2 cm3; after that the animals were observed up to 12 months. The excised tumors still contained and expressed the transfected ras oncogene as proved by Southern blot analysis and antibody staining using anti-ras p21. In contrast to most previous work on ras-transfected tumorigenic cells the R1H-ras tumors did not acquire invasive growth potential or increased metastatic capacity.