Overexpression of c-K-ras, c-N-ras and transforming growth factor {beta} co-segregate with tumorigenicity in morphologically transformed C3H 10T1/2 cell lines

Morphologic transformation and tumorigenicity are separate cellularphenotypes in transformed 1OT1/2 cells. We have investigatedthe levels of expression of genes for c-myc, c-H-ras, c-K-ras,c-N-ras, TGFß and Rb in 42 morphologically transformed1OT1/2 cell lines, in an attempt to define the molecular mechanisntsgoverning morphologic transformation and tumorigenicity in the1OT1/2 cell system. The 101 1/2 cell lines investigated generallyoverexpressed mRNAs for c-myc, c-H-ras, and TGFß relativeto the levels expressed by wild- type 10T1 cells (levels ofexpression >1.5-fold that of wild- type 1011/2 cells). Incontrast, only half of these cell lines overexpressed mRNAsfor c-N-ras and/or Rb relative to wild- type 1OT1/2 cells, andonly 25%; overexpressed c-K-ras mRNA. The mean levels of mRNAexpression for each of c-K-ras, c-N-ras and TGFß genesin tumorigenic cell lines were significantly greater than themean levels of expression in non-tumorigenic cell lines, suggestingan association between twnorigenicity and the levels of expressionof these specific genes. In contrast, levels of expression forc-myc, c-H-ras and Rb genes were not correlated with tumorigenicity.Cell lines that coexpressed high levels of c-K-ras, c-N-rasand TGFß genes were likely to be tumorigenic (11/12cell lines were tumorigenic), whereas cell lines that coexpressedlow levels of these genes were unlikely to be tumorigenlc (1/10cell lines were tumorigenic). High expression of TGFßwas sufficient for tumorigenicity in the absence of high levelsof expression of c-K-ras and c-N-ras (5/5 cell lines were tumorigenic).Elevated expression of either c-K-ras or c-N-ras alone was Insufficientfor tumorlgenlclty, however, coordinate overexpresslon of bothc-K-ras and c-ras was associated with tumorigenicity irrespectiveof the expression status for TGFß (13/15 cell lineswere tuinorigenic). These results suggest that overexpresslonof c-ras, c-H-ras and TGFß are conunonly associatedwith, and possibly mechanistically related to, the process ofmorphologic transformation in 1OTI/2 cells. In addition, theseresults suggest that progression from morphologic transformationto tumorigenicity in 1011/2 cell lines is frequently accompaniedby overexpression of c-K-ras and c-N-ras, and by enhancementof the level of overexpresslon of TGFß     

Transformation of immortalized woodchuck hepatic cell lines with the c-Ha-ras proto-oncogene

Woodchuck hepatocytes were immortalized with the simian virus40 T antigen (SV40 T-ag) oncogene and utilized in an oncogenictransformation assay. Transfection of these cell lines withan activated c-Ha-ras oncogene (EJ6.6) resulted in the transformationof cells to a phenotype characterized by anchorage-independentgrowth in soft agar. Colonies of transformed cells were subclonedand up to 80% were positive for oncoprotein expression detectedby immunoblotand Northern blot procedures. When compared withthe parental cell lines, ras-transformed derivatives were alteredboth morphologically and in growth rate. The tumorigenic potentialof c-Ha-ras transformed cells was demonstrated in severe combinedimmunodeficient (SCID) mice. There was a latency period of 1to 4 weeks before tumors were detectable and a period of over7 weeks was required for tumors to reach a diameter of 1 cm.Histologically, tumorsderived from cell lines fully transformedby the SV40 T-ag had the appearance of well differentiated hepatocellularcarcinoma (HCC) while tumors derived from c-Ha-ras transformedcell lines had the appearance of poorly differentiated HCC.The capacity to induce oncogenic transformation events in immortalizedwoodchuck hepatic cell lines should provide the opportunityto study the cooperative effects of hepadnaviral genes in hepatocarcinogenesisin vitro.     

Activated ras oncogene and specifically acquired resistance to cisplatin in human mammary epithelial cells: induction of DNA cross-links and their repair

A human non-malignant mammary epithelial cell line, HBL100,and the ras-transformed HBL100/ras1 cell line were examinedfor their sensitivity to cis-diamminedichloroplatinum(II) (cisplatin).The clonogenic cell survival assay showed that HBL100/ras1 exhibiteda 2.7-fold increased resistance compared to the parental HBL100cell line. The responses to other agents interacting with DNA,such as mitomycin C, 8-methoxypsoralen plus UVA or doxorubicin,were very similar in both cell lines. The same is true for ionizingradiation (Alapetite et al., Int J. Radiat. Biol., 59, 385–396,1991). In other words, the mechanism of acquired resistancein HBL100 appears to be limited to cisplatin. No differencewas observed between the two cell lines in cisplatin uptakeas determined by atomic absorption spectrometry. Alkaline elutionshowed that less interstrand cross-links were formed by thisdrug in the resistant HBL100/ras1 cells compared to HBL100 and,moreover, the removal of these adducts was clearly more efficientin the former cell line. This was confirmed by an in vitro excisionrepair assay which revealed a 2.2-fold increase in DNA repairactivity in the extracts from HBLlOO/ras1 versus HBL100 cells.It is concluded that the transformation of human epithelialHBL100 cells by the ras gene resulted in an acquired resistanceapparently limited to cisplatin, a feature associated with areduced proportion of induced interstrand cross-links and ahigher efficiency in their removal. The mechanism of involvementof the ras gene product in this process is still a matter ofspeculation.     

Activation of H-ras oncogene in 3-methylcholanthrene-transformed human cell line

DNA prepared from the 3-methylcholanthrene (3MC)-transformedhuman 312H cell line induced foci on NIH/3T3 cells, whereasDNAs prepared from 7,12-dimethylbenz[a]-anthracene-transformedand the dimethylsulfoxide control 312H cell lines failed toinduce foci. The transformed gene from the 3MC-transformed 312Hcells was identified as an activated form of the human cellulartransforming H-ras oncogene. Analysis of the ras oncogene p21product in this transformant by immunoprecipitation and gelelectrophoresis suggested that this gene was activated by mutationin the 61st codon. These findings demonstrate that activationof a member of the ras gene family can occur in a chemicallytransformed human cell line.     

Growth inhibition of rat liver epithelial tumor cells by monoterpenes does not involve Ras plasma membrane association

The role of altered ras oncoprotein (Ras) farnesylatlon andmembrane association in the growth inhibitory effects of severalmonoterpenes (limonene, perillic acid, perilyl alcohol, menthol,pinene and cineole) was investigated in rat liver epithelialcells. All of the above compounds except cineole inhibited thegrowth of viral Ha-ras-transformed rat liver epithelial cells(WB-ras cells) at concentrations of 0.25–2.5 mM. Thesecells, however, were not necessarily more sensitive to thesecompounds compared to non-transformed and viral raf-transformedrat liver epithelial cells. Growth inhibition by limonene, perillicacid and pinene was only partially restored (20–50%) bysupplementing the culture medium with 2 mM mevalonic acid. Westernblot analyses of cytosolic and membranous fractions of WB-rascells treated with monoterpenes indicated no change in Ras distribution.In contrast, lovastatin, a potent inhibitor of 3-hydroxy-3-methyl-glutarylcoenzyme A reductase and Ras farnesylation, specifically reducedWB-ras cell growth and increased cytosolic levels of Ras. Thus,monoterpene-induced growth inhibition of rat liver epithelialcells was dissimilar to lovastatin and did not apear to involvealtered Ras plasma membrane association.     

Transformation of human breast epithelial cells by chemical carcinogens

The present study was carried out to determine whether humanbreast epithelial cells (HBEC) are transformed by chemicalsthat have been proven to be carcinogenic in other model systems.A spontaneously immortalized human breast epithelial cell line,MCF-10F, was treated with dimethylbenz[a]anthracene (DMBA),benzo[a]pyrene (B[a]P), N-methyl-N-nitrosoguanidine (MNNG) orN-methyl-N-nitrosourea (NMU) for 24 h. MCF-7 and T24 malignantcell lines were used as positive controls. All the carcinogensinduced alterations in both cell morphology and pattern of growth,increased growth rate and anchorage-independent growth in agar—methocel,which became evident by the 8th to 10th passages post-treatment,at     

Transformation of rat liver epithelial cells with v-H-ras or v-raf causes expression of MDR-1, glutathione-S-transferase-P and increased resistance to cytotoxic chemicals

We have examined the relationship between transformation andmultidrug resistance by employing the v-H-ras or v-raf oncogenesto transform rat liver epithelial (RLE) cells in vitro. Thedataindicate that transformation of RLE cells with v-H-ras orv-raf results in increased resistance to the cytotoxins adriamycin,vinblastine and 2-acetylaminofluorene. This multidrug resistanceis accompanied by increasing expression of P-glycoprotein (MDR-1)and glutathione-S transferase P (GST-P). Thus, neoplastic transformationofRLE cells with v-raf or v-H-ras, independently of chemicalexposure, results in multidrug resistance.     

DNA contents and chromosomes of clonal lines of transformed rat liver epithelial cells and of cells from their derived tumors

Clonal lines of transformed rat liver epithelial cells, derivedfrom a single population of cloned diploid rat liver epithelial(stem-like) cell line (WB-F344) by exposure in vitro to N methyl-N'-nitro-N-nitrosoguanidine(MNNG), produce hepatocellular carcinomas, hepatoblastomas andadeno carcinomas in syngeneic rats (Tsao and Grisham, Am. J.Pathol., 127, 168–181, 1987). In this study we show thatthese clonal lines demonstrate near-diploid (GN clones) or near-triploid(GP clones) aneuploidy and the universal occurrence of non-randomchromosomal abnormalities. Marker chromo somes that involvedfour autosomes-a non-reciprocal translocation involving chromosomes1 and 7 (t1q43;7q34), and addition of DNA of unknown originto the pericentro meric regions of chromosomes 4 and 10—occurredin all of the cells of all transformed clones and in the cellsof tumors that grew from them. New marker chromosomes involvingthe same regions of chromosomes 4 and 7 were found in severalcell lines established from independent tumors. The preservationof marker chromosomes in tumor cells in the face of random lossand gain of other chromosomes suggests that these non-randomaberrations were necessary for tumor formation. The presenceof marker chromosomes was associated with increased expressionof the c-myc gene (located at q34 on chromosome 7), the c-H-rasgene (located at q41–43 on chromosome 1) and the c-K-rasand TGF     

Transformation related expression of glutathione-S-transferase P in rat liver cells

A neutral isoenzyme of glutathione transferase designated glutathione-S-transferase7-7, but also referred to as glutathione-S-transferase P (GST-P)is absent from adult rat liver hepatocytes, but expressed ata very early stage in chemically induced in vivo hepatocarcinogenesis.The expression of this enzyme in a range of aflatoxin B₁ (AFB₁)associated hepatocarcinogenic systems has been examined, includingin vivo induced preneoplastic and neoplastic liver tissue, celllines derived from hepatomas, primary hepatocytes in cultureand an immortalized rat liver cell line before and after transformationin vitro either by transfection with ras oncogenes or by treatmentwith metabolically activated AFB₁. Analyses of total cytosolproteins using a polyclonal antibody to GST-P did not detectthe presence of GST-P protein in cytosols from control or regeneratingliver. A low level of expression was detected in the immortalized,non-transformed epithelial cell line, but a greatly inducedlevel occurred subsequent to transformation of these cells byeither of the two techniques used. High levels of the proteinwere detected in in vivo induced preneoplastic and neoplastictissues, and in the cell lines derived from them. Total RNAfractions isolated from the various cells or tissues, when examinedwith a cDNA probe for GST-P mRNA, showed it to be absent fromcontrol and regenerating rat liver. It was present at low levelsin the untransformed cell line and primary hepatocytes after48 h in culture, but present at greatest abundance in the invivo and in vitro transformed cells. The results indicate thatthe highest elevation in expression of this protein is associatedwith the stage of definitive malignant transformation in invitro carcinogenesis, and this could have relevance in definingcomparable events in the in vivo multistage sequence.     

Detection of distinct transforming genes in X-ray induced tumors

DNAs from mouse skin tumors (papillomas, squamous cell carcinomas,basal cell carcinomas and pilomatrixomas) initiated with X-irradiationand promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA)demonstrated dominant transforming activity by the productionof transformed foci in the mouse recipient tine, NIH3T3. Dominanttransforming activity was not found in DNA isolated from normalmouse epidermis or from the corresponding liver. The NIH3T3transformants induced with squamous cell carcinoma DNA grewin soft agar and formed tumors in nude mice. Southern blot analysisof primary NIH3T3 transformant DNAs carrying oncogenes fromradiation-initiated squamous cell carcinomas indicated thatthe oncogenes responsible for the transformation of the recipientcells were not Ha-ras, Ki-ras or N-ras genes, nor were theyerbB, B-lym, met, neu or raf. The data presented indicate thatDNAs from radiation-initiated mouse skin tumors contain dominanttransforming genes that are detectable by DNA-mediated genetransfer. The oncogene sequences activated in these radiation-initiatedtumors are distinct non-ras transforming genes.     

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells

In order to study the relationship between altered gap junctionalintercellular communication (GJIC) and induction of cell transformationby oncogenes, we transfected six viral oncogenes into BALB/c3T3A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyomamiddle T (PyMT) genes grew in soft agar and formed distincttransformed foci in the absence or presence of a vast excessof non-transfected cells. On the other hand, those with v-myc,v-fos or polyoma large T (PyLT) genes expressed less distinctlytransformed phenotypes (less transformed morphology, highersaturation density than non-transfected counterparts and lessgrowth in soft agar), and did not form distinct foci in coculturewith non-transformed cells. When their homologous GJIC capacitieswere examined by the microinjection/dye transfer assay, no decreasein GJIC was observed in any of the v-onc-transformed cells.Non-transformed and all v-onc-transformed cell lines expressedsimilar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformedcells, but not v-ras-, v-src- and PyMT-transformed cells wereable to communicate heterologously with non-transformed cells.Tumor promoting phorbol esters strongly inhibited GJIC of non-transformedand all v-onc-transformed BALB/c3T3 cell lines. In coculturesof v-myc-, v-fos- or PyLT-transformed cells with non-transformedBALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate(TPA) increased the number of transformed foci. However, whenthese v-onc-transformed cells were co-cultured with non-transformedBALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence,as if TPA had been added), no morphologically transformed fociappeared. These results suggest that factors other than GJICare involved in the suppression of oncogene-transformed cellsby surrounding normal counterparts.     

Down-regulation of riα subunit of camp-dependent protein kinase induces growth inhibition of human mammary epithelial cells transformed by c-ha-ras and c-erbb-2 proto-oncogenes

MCF- 10A is a spontaneously immortalized, non-transformed human mammary epithelial cell line. We have recently obtained MCF- 10A clones (MCF- 1OA HE cells) that are transformed following over-expression of both a human point-mutated c-Ha-ras and the c-erbB-2 proto-oncogenes. Two isoforms of the cAMP-dependent protein kinase (cAK) have been described in mammalian cells. Enhanced levels of type-I cAK (cAKI) are generally found in tumor cells. To determine whether inhibition of cAKl expression may interfere with ras and erbB-2 oncogene-induced transformation of human mammary epithelial cells, we have tested the effects of 2 agents that specifically down-regulate cAKI, such as 8-chloro-cAMP and an anti-sense oligodeoxynucleotide targeted against the R1α regulatory subunit of cAKl on MCF-10A HE cells. Treatment of MCF-10A HE cells with 8-chloro-cAMP induces a dose-dependent growth inhibition under both monolayer and soft-agar growth conditions, that is correlated with an accumulation of MCF-10A HE cells in G₀/G, phases of the cell cycle and a reduction of the number of cells in S phase. In contrast, 8-chloro-cAMP has no effect on MCF-10A cell growth. Furthermore, 8-chloro-cAMP treatment of MCF-10A HE cells induces a 4- to 6-fold reduction in p185erbB-2 expression and brings p21 ras expression to levels comparable to those found in MCF-10A cells. Treatment of MCF-10A HE cells with an Rlα anti-sense oligodeoxynucleotide determines a comparable inhibition of both anchorage-dependent and anchorage-independent cell growth. Our results suggest that cAKl may act as a mediator of ras and erbB-2 oncogene action in human breast cells and that interference with cAKl action provides a potential tool for inhibiting the growth-promoting effects of these oncogenes.     

Association of deficient DNA repair during G2 phase with progression from benign to malignant state in a line of human skin keratinocytes transfected with ras oncogene

Human skin keratinocytes after malignant neoplastic transformationby infection with Kirsten murine sarcoma virus (KiMSV) or transfectionwith pSV² ras (containing an activated c-Ha-ras oncogene) showeda DNA repair deficiency(ies). The repair deficiency was manifestas an abnormally high frequency of chromatid breaks and gapspersisting after X-ray-induced DNA damage inflicted during theG₂ phase of the cell cycle. Non-tumorigenic control cells atthat time were clearly repair-efficient. By analyzing benignand malignant tumorigenic HaCaT-ras clones, we could excluderas p21 oncoprotein expression as the causal mechanism for repairdeficiency, since both clone types expressed similar levelsof the mutated protein and only the malignant tumorigenlc cellsshowed repair deficiency. The results suggest that mutated p21ras provided the human keratinocytes with a growth advantagein vivo (benign tumor growth), but acquisition of repair deficiencyis required for progression from benign to malignant state.     

Nickel(II) induces alterations in EGF- and TGF-{beta}1-mediated growth control during malignant transformation of human kidney epithelial cells

We have previously described immortalization of normal humankidney epithelial cells by nickel(H) and the subsequent tumorigenicconversion by v-Ha-ras transfection. We report here that nickel(II)induces alterations in growth regulatory control. Normal humankidney epithelial cells (NHKE) were growth inhibited by transforminggrowth factor ß (TGF-ß). This effect wasaborgated in both the immortalized (IHKE) and transformed (THKE)cells. (NHKE) expressed 4700 high-affinity binding sites/cellfor TGF-ß₁. IHKE and THKE showed reduced binding of47% and 44% relative to NHKE respectively. On the other hand,expression of epidermal growth factor (EGF) receptors was elevatedin IHKE (260%) and THKE (236%) relative to NHKE, which expressed1.5 x 10⁵ receptors/cell. Preincubation of IHKE and THKE withTGF-ß₁ resulted in reduced EGF binding, whereas thisbinding was unaltered in NHKE. Exposure of human kidney epithelialcells to EGF led to tyrosine phosphorylation of the EGF receptorand other cellular proteins in the mol. wt range from 42 to>300 kDa. The level of receptor phosphorylation induced byEGF reflested receptor expression. Tyrosine phosphorylated proteinsappear to be identical in all three cell lines, and reach phosphorylationmaxima independently of EGF receptor expression. These studiesindicate that nickel carcinogenesis may involve changes in setsof genes important in normal growth regulation.     

High mammary carcinogenicity of neutron irradiation in rats and its promotion by prolactin

The present study was undertaken to assess the mammary carcinogenic effect of low doses of fission and thermal neutrons in female W/Fu rats. Only 2 of 62 (3.2%) rats exposed to various doses of fission radiation alone developed mammary tumors (MT) in a 12-month observation period, whereas 21 of 63 (33.3%) similarly irradiated rats developed MT if further treated with prolactin; the lowest effective dose was 4.1 rad, which contained only 1.7 rad of fission spectrum neutrons with an average energy of 2.0 MeV. The long survival of radiation-initiated potentially malignant cells was suggested by the observation that excess numbers of MT developed in irradiated rats in whom prolactin treatment was started as late as 12 months after irradiation. The negligible contribution of gamma-rays, one component of the reactor radiations, to the rat mammary carcinogenesis was proven by a simulation experiment with 60Co gamma-rays. Thermal neutrons with an average energy of 0.025 eV were less effective than fission neutrons. The rat mammary carcinogenic effects of 180 kVp X-rays, 14.1 MeV fast neutrons, 0.025 eV thermal neutrons and 2.0 MeV fission neutrons were estimated in such a way as to compare the dose of each radiation that gave an MT incidence of 40% of that in irradiated, prolactin-treated rats. The efficiency of fission spectrum neutrons is much higher than those of other radiations; the relative biological effectiveness (RBE) of fission spectrum neutrons was 17.8 against X-rays. Based on these findings, the relevance of animal data to tumor induction in atomic bomb survivors is discussed.     

Expression and function of connexin in normal and transformed human keratinocytes in culture

We have studied the gap junctional intercellular communication(GJIC) of immortalized and tumourigenic human keratinocyte celllines and of a spontaneously immortalized non-tumourigenic anda highly differentiating keratinocyte cell line (HaCaT) as thecontrol. In homologous cultures, the GJIC capacity of five squamouscell carcinoma-derived cell lines was 1–27% that of theHaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenicpotential and an SV40 DNA-immortalized cell line had markedlyreduced GJIC capacities. Northern analysis and immunohisto-chemistryshowed that connexin (Cx) 43 is the major gap junction proteinexpressed in the communicating cells. They do not express Cx26 or 32. The low or absent communication observed in certaincell lines was due in some to a lack of Cx 43 gene expression,but in others to aberrant localization of the gap junction protein.GJIC of these cell lines, as well as that of primary normalhuman epidermal keratinocytes, was susceptible to 12-O-tetra-decanoylphorbol-13-acetate-mediatedinhibition. Moreover, GJIC of HaCaT cells and their tumourigenicderivatives is Ca²⁺-dependent. These results, when comparedwith those previously obtained for mouse keratinocyte cell lines,reveal that GJIC of human keratinocytes was correlated to thedegree of differentiation and is controlled in a similar wayto that of murine keratinocytes. Aberrant GJIC seems to be acommon feature of human and murine skin carcinogenesis.     

Correlation of (6-4)photoproduct formation with transforming mutations in UV-irradiated Ha-ras

Sites and types of mutations in relation to the amount of cyclobutanepyrimidine dimers (CPDs) and (6–4)-photoproducts in UV-irradiatednormal Ha-ras sequences were investigated. Mouse BALB/C 3T3cells were transfected with UV-irradiated pYN-mHras plasmidscontaining mouse normal Ha-ras sequences, and transformed focideveloped. Direct DNA sequencing of the Ha-ras retrieved fromthe foci revealed that most mutations (23/24, 96%) took placeat dipyrimidine sequences, and the CT transition at the 3'-cytosinein 5'-TC or 5'-CC sequences was predominant (17/24, 71%) incodons 12, 13 and 60. In codon 61, where 5'-TC or 5'-CC is absent,two mutations were found at the 5'-TT sequence. More (6–4)photoproductswere produced than CPDs in codons 12, 13 and 60, and more CPDswere produced than (6–4)photoproducts in codon 61. Theseresults suggest that (6–4)photoproducts are the majorlesion leading to the mutations in the mouse Ha-ras sequenceand subsequent transformation of BALB/C 3T3 cells.