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.     

Overexpression of ha-ras oncogene transforms rodent fibroblasts with low-frequency but not human-diploid fibroblasts

Activated Ha-ras oncogenes were introduced into early passage normal human adult dermal fibroblasts, 149BR and established mouse Swiss 3T3 fibroblasts by retroviral-mediated gene transfer or by DNA transfection, respectively. The presence and expression of Ha-ras oncogenes was followed by Southern and Northern blotting and immunoprecipitation. Overexpression of the human mutant c-Ha-ras1 T24 oncogene in mouse cells induced morphological transformation, focus formation and growth in soft agar with low frequency. Tumourigenicity studies showed that the malignant transformation of these cells by p21Ha-ras T24 oncoprotein was concentration-dependent and it required additional events suggesting that in vitro establishment is not a sufficient prerequisite for tumourigenic conversion by activated Ha-ras oncogenes. In contrast, constitutive expression of the viral Ha-ras oncogene in human diploid fibroblasts failed to immortalise or transform them. All ras-expressing human cells remained flat, anchorage-dependent and non-tumourigenic suggesting that these cells are resistant to transformation by activated oncogenes. The role of Ha-ras oncogenes in the transformation of mammalian cells in discussed.     

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.     

Immunohistochemical analysis of the ras p21 oncoprotein in Hashimoto's thyroiditis.

We studied the ras oncogene expression using immunohistochemical detection of p21 oncoprotein in paraffin-embedded tissue sections or fine needle aspiration (FNA) material from 25 patients with Hashimoto's thyroiditis and 12 healthy individuals. We also investigated the presence of this protein in the lymphocytes of thyroid gland, as well as in peripheral blood lymphocytes. We found increased expression of p21 oncoprotein by thyroid epithelial cells in 22 patients (intensity of staining ++), whereas we observed negative or slightly positive in 9 and 3 out of 12 normal controls, respectively (intensity of staining, - or +/-). We also detected p21 oncoprotein in moderate amounts in patients' intrathyroid lymphocytes (intensity of staining +), but peripheral blood lymphocytes did not present any staining result. Our findings provided evidence that epithelial cells, as well as lymphocytes infiltrating the thyroid gland, are probably "activated" in Hashimoto's thyroiditis. The significance of this "activation" in thyroid tumorigenesis remains unknown.     

Melittin resistance: a counterselection for ras transformation.

The prevalence of activated ras oncogenes in human primary tumors suggests a central role for this oncogene in human cancer. Despite its ubiquitous distribution, the biochemical role of the oncogene remains unclear, and hence attempts to control its activity have been frustrated. This study demonstrates the ability of melittin, a 26 amino acid, amphipathic peptide from bee venom, to specifically select against cells in culture that express high levels of the ras oncogene. Acquisition of resistance to increasing concentrations of melittin is accompanied by corresponding decreases in the levels of expression of the ras oncoprotein and the number of copies of the ras gene. This results in a concomitant reversion of transformed cells to a normal morphology in a strict dose-dependent manner. Melittin is a known activator of cellular phospholipase A2 (PLA2), and these results suggest an interrelationship between ras and PLA2. In addition these studies indicate that melittin preferentially hyperactivates PLA2 in ras oncogene-transformed cells, resulting in their selective destruction.     

Elevation of glucose transporter, c-myc, and transin RNA levels by Ha-rasT24 is independent of its effect on the cell cycle

Elevation of the steady-state mRNA levels of glucose transporter and c-myc are among the earliest changes in gene expression observed after Ha-rasT24 stimulation of Rat-1 fibroblasts to enter the cell cycle. Since the expression of these genes may be the result of either increased cell proliferation or a specific response to rasT24, we evaluated the expression of glucose transporter and c-myc and their induction during the cell cycle in both parental Rat-1 cells and cell lines bearing a metallothionein rasT24 fusion gene (MTrasT24). We showed that, although levels of glucose transporter and c-myc mRNAs in Rat-1 cells underwent a transient increase within hours of the addition of serum, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate to quiescent (G0) cells, the levels of glucose transporter and c-myc mRNA otherwise remained constant throughout the normal cell cycle. In cells carrying MTrasT24 (MR5 cells), induction of rasT24 expression by ZnSO4 led to a rapid induction of glucose transporter and c-myc mRNA expression in both quiescent (density-arrested) and G1/S-synchronized (aphidicolin-blocked) cells. These increases exceeded the constitutive levels expressed in rapidly proliferating Rat-1 cells, indicating that the ras oncogene has an effect on these genes that is independent of growth status. In addition, the transin gene, which is not expressed in proliferating Rat-1 cells in the continuous presence of serum growth factors, was also induced after increased expression of the mutant ras gene. These results suggest that the induction of glucose transporter, c-myc, and transin is the direct result of rasT24-mediated alterations in cellular gene expression and is distinct from normal cell-cycle events.     

Expression of the c-Ha-ras oncogene in mouse NIH 3T3 cells induces resistance to cisplatin.

The effect of expression of the c-Ha-ras oncogene on cisplatin (DDP) sensitivity was examined in murine NIH 3T3 cells transfected with the dexamethasone (DEX)-inducible mouse mammary tumor virus promoter linked to an activated c-Ha-ras gene [LTR H-ras(A) cells]. Treatment of these cells with 5 microM DEX for 24 h induced c-Ha-ras expression and produced an 8.2 +/- 1.3-fold (SD) increase in DDP resistance as quantitated by clonogenic assay. Induction of the c-Ha-ras oncogene reduced DDP accumulation by 40% and intrastrand adduct formation by 17%. In nontransfected wild-type NIH 3T3 cells, DEX did not induce DDP resistance nor did it decrease DDP accumulation. Induction of c-Ha-ras expression did not alter cellular glutathione content or the activity of glutathione-S-transferase in the LTR H-ras(A) cells. DEX increased cellular metallothionein content by 1.6-fold in NIH 3T3 cells and 3.3-fold in LTR H-ras(A) cells. We conclude that DEX-induced overexpression of a mutant c-Ha-ras gene confers DDP resistance and that this resistance is associated with an impairment of cellular drug accumulation and an increase in metallothionein content.     

Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry

The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation. We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content. Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method. Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide). The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia. The p21 ras oncoprotein was found in all phases of cell cycle. The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells. Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6] and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.     

Regulation of p21/ras protein expression by diallyl sulfide in DMBA induced neoplastic changes in mouse skin

Diallyl sulfide (DAS), a naturally occurring organosulfide, present in garlic, is known to possess pleiotropic biological effects. DAS is known to inhibit chemically induced tumors in a number of animal models. The chemopreventive properties of DAS seem to occur through a number of mechanisms, but its role on primary events on oncogenic activation is not well understood. In the present study, we demonstrated the modulatory effect of DAS on the expression of H-ras gene product, p21/ras protein as one of the mechanisms of its chemopreventive action in chemically induced mouse skin tumors. Our results showed that DAS administration leads to modulation of the DMBA-induced levels of p21/ras oncoprotein as early as 24h after the DMBA application, suggesting down-regulation of the p21/ras by DAS. Furthermore, the modulatory effects of DAS were also evident in DMBA-induced mouse skin tumors. DAS administration led to increase in the levels of cytosolic p21/ras and decrease in the levels of p21/ras in membrane fractions. DAS administration was also found to down regulate the DMBA-induced H-ras mRNA level in mouse skin tumors. The immunohistochemical staining of the skin/tumor showed 55.82 and 46.86% decrease in the area positive for p21/ras expression levels in DAS pre- and post-supplemented groups, respectively. Flow-cytometric analysis, further confirms our results as indicated by a shift in the mean fluorescence intensity (MFI) towards lower fluorescence in DAS administered groups in comparison to the DMBA treated group. Thus, one mechanism of the growth inhibitory properties of DAS is through the suppression of development of tumors that harbor ras mutations by inhibiting the membrane association of oncogenic p21/ras protein.     

Localization of oncoprotein P21ras in the human liver cancer

Using the human liver cancer DNA transfected NIH/3T3 cell line, the human N-ras oncogene and the over expression of the oncoprotein P21ras was demonstrated, BALB/C mice were immunized. The spleen cells from the immunized mice were fused with SP2/0 myeloma cells. After the HAT medium selection and screening, two hybridoma cell lines, SCI-Oncogema 1 and 2, were established. In the immunoprecipitation test, the molecular weight of the protein reacting to Oncogema 1 was 21,000. This M.W 21,000 protein possessed the capability to bind with GTP, i.e. the character of P21ras. These data indicate that the Oncogema 1 is the monoclonal antibody against P21ras. Using Oncogema 1, specimens from 6 liver cancer patients were studied by immunopathology. With ABC stain, it was observed that the malignant cells in all the samples showed dark staining; the P21ras revealed over expression. Although the staining was heterogeneous, it implied that the ras oncogene was involved in the carcinogenesis of these six samples. No over expression was seen in the normal liver cells even in those around the cancerous lesion. However, dysplastic cells were moderately stained which means that the ras oncogene was activated and P21ras over expressed in these cells. The results suggest that the ras oncogene and P21ras play an important role in the early stage of liver cancer carcinogenesis.     

Partial down-regulation of protein kinase C in C3H 10T 1/2 mouse fibroblasts transfected with the human Ha-ras oncogene

Biochemical and immunological comparison of mouse C3H 10T 1/2 fibroblasts and C3H 10T 1/2 fibroblasts transfected with human activated Ha-ras oncogene indicated significantly lower levels of protein kinase C (PKC) activity and protein in the ras-transfected cells. This effect was observed in three clonal cell lines transfected with an activated ras oncogene. Cytosolic extracts of the ras-transfected cells contained calcium-activated, phospholipid-dependent protein kinase (PKC) activity at 61% of the level of activity present in C3H 10T 1/2 cells. A similarly decreased level of phorbol ester-binding activity was observed in these cells. Analysis of the subcellular distribution of PKC activity in cells failed to indicate significant differences between these cell lines. Immunoblots showed a lower abundance of the Mr 80,000 PKC in ras-transfected cell homogenates and extracts compared to C3H 10T 1/2 cells. Both C3H 10T 1/2 cells and cells transfected with ras expressed only one of the PKC isozymes as resolved by hydroxylapatite chromatography demonstrating that ras transfection of cells did not induce expression of alternative PKC isozymes. These observations indicate that PKC was partially down-regulated in ras-transfected cells, perhaps resulting from constitutively elevated levels of products of phosphatidylinositol-4,5-bisphosphate hydrolysis. Although C3H 10T 1/2 cells were previously shown to be distinct from NIH 3T3 cells in their sensitivity to transformation by the T24-ras oncogene, ras transformation appears to partially down-regulate PKC in C3H 10T 1/2 cells in a manner identical to that for ras-transformed NIH 3T3 cells. This indicates that down-regulation of PKC directly results from the expression of an activated ras oncogene independently of cellular sensitivity to transformation by expression of ras. The common action of ras transformation and phorbol esters to down-regulate PKC provides a possible mechanism for synergism during multistage carcinogenesis.     

Expression of transforming growth factor alpha (TGF alpha) in breast cancer

Transforming growth factor alpha (TGF alpha) is one growth factor that has been circumstantially implicated in regulating the autocrine growth of breast cancer cells. Expression of TGF alpha can be modulated by activated cellular protooncogenes such as ras and by estrogens. For example, the epidermal growth factor (EGF)-responsive normal NOG-8 mouse and human MCF-10A mammary epithelial cell lines can be transformed with either a point-mutated c-Ha-ras protooncogene or with a normal or point-mutated c-neu (erbB-2) protooncogene. In ras transformed NOG-8 and MCF-10A cells but not in neu transformed cells there is a loss in or an attenuated response to the mitogenic effects of EGF. This response may be due in part to an enhanced production of endogenous TGF alpha that is coordinately and temporally linked to the expression of the activated ras gene and to the acquisition of transformation-associated properties in these cells. TGF alpha mRNA and TGF alpha protein can also be detected in approximately 50-70% of primary human breast tumors. In addition, approximately 2- to 3-fold higher levels of biologically active and immunoreactive TGF alpha can also be detected in the pleural effusions from breast cancer patients as compared with the TGF alpha levels in the serous effusions of noncancer patients. Over-expression of a full-length TGF alpha cDNA in NOG-8 and MCF-10A cells is capable of transforming these cells. Finally, expression of TGF alpha mRNA and production of biologically active TGF alpha protein is also found in normal rodent and human mammary epithelial cells.     

Reversible suppression by nalidixic acid of anchorage-independent growth of mouse cells transformed by 3-methylcholanthrene or an activated c-Ha-ras gene

Effects of nalidixic acid and its derivatives were investigated on mouse cells transformed by methylcholanthrene or an activated c-Ha-ras oncogene. Our findings were as follows. Nalidixic acid preferentially suppressed growth in soft agar of transformed Balb/3T3 mouse cells induced by methylcholanthrene. The suppressive effect of nalidixic acid on growth in soft agar was reversible. Nalidixic acid reversibly reduced saturation density of these transformed cells. Oxolinic acid and pipemidic acid, which are derivatives of nalidixic acid, were less effective than nalidixic acid in suppressing growth in soft agar. Nalidixic acid suppressed growth in soft agar of NIH/3T3 mouse cells transformed by an activated c-Ha-ras, without affecting the amount of ras p21 proteins as detected by an immunoblotting analysis using a monoclonal antibody. These results show that nalidixic acid reversibly suppressed the expression of transformed phenotypes that were already being expressed.