Tumour perfusion and associated physiology: Characterization and significance for hyperthermia

Mouse embryo cells (C3H 10T1/2) were transfected with a plasmid (pAL8A) containing the HRas oncogene and neomycin resistance gene. Five transfected cell clones were isolated and established as cell lines, and these showed neomycin resistance. Two of these cell lines expressed a normal morphology while three showed a transformed morphology. Four of the cell lines produced tumours in nude mice. Flow cytometry measurements showed that exponentially growing cell cultures of the five transfected cell lines had the same cell cycle distribution as the normal parental cell line. The sensitivity to hyperthermia of the five transfected cell lines was the same as that of the normal cell line for temperatures ranging from 42.0 to 45.0°C. Thus in these studies we found no difference in the thermal sensitivity of normal and malignant cells transfected by the Hras oncogene.     

Thermal response of oncogene-transfected rat cells

Rat embryo cells or Rat-1 fibroblasts were transfected with either an activated c-myc or a c-Ha-ras from the T24/EJ bladder carcinoma, or they were cotransfected with both. A gene conferring neomycin or hygromycin resistance was also cotransfected so that independent cell lines could be selected by growth in medium containing the antibiotic. Certain isolates from cells transfected with only one type of oncogene were further transformed by exposure to 600 cGy of 250-kVp X-rays. Successful transfection and transformation were characterized by altered morphology, increased plating efficiency, shorter doubling time, longer life span, foci formation, anchorage-independent growth, and Southern and Northern hybridization analysis. The thermal response of these cells at different stages of oncogenic transformation was examined by exposing exponentially growing cells to 45 degrees C for 0 to 45 min and measuring cellular survivals using colony formation assay. We found that cells transfected with myc oncogene, singly or in combination with ras, were more sensitive to thermal stress. Aside from that, the cells' thermal sensitivity was not affected by the degree or the nature of transformation.     

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.     

lacZ-neoR transfected glioma cells in syngeneic rats: growth pattern and characterization of the host immune response against cells transplanted inside and outside the CNS

The rat glioma cell lines BT4C and BT4Cn were stably transfected with the bacterial lacZ-neomycin resistance (neoR) gene construct. Both transfected (BT4ClacZ and BT4CnlacZ) and untransfected cell lines were injected intracerebrally and subcutaneously into rats. Survival time, morphology, growth rate and immunological properties of the tumors were studied. Survival time was significantly prolonged after intracerebral implantation of the transfected cell lines. No similar response was found in nude rats, indicating an immunological response towards the lacZ-neoR-transfected cells in immunocompetent animals. Morphological observations showed that the lacZ-neoR-transfected gliomas were smaller and had a distinct boundary with the normal brain tissue, whereas the parental cell lines revealed a more diffuse growth pattern. Immunostaining showed a higher proportion of immunocompetent cells infiltrating the lacZ-neoR-transfected tumors. After s.c. injection, the lacZ-neoR-transfected BT4C cell line had a prolonged lag phase before assuming a growth rate similar to that of the parental cells. The BT4CnvlacZ tumors initially grew fastest, but then disappeared within 3 weeks. A similar response was observed with mock-transfected tumor cells. A (3)HTdR-incorporation assay on spleen cells from rats transplanted s.c. with BT4CnvlacZ cells showed a 10-fold increase in cell activation as compared with rats with BT4Cn tumors. A humoral response towards the transfected cells was verified by Western-blot analyses.     

Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Early cell motility changes associated with an increase in metastatic ability in rat prostatic cancer cells transfected with the v-Harvey-ras oncogene

The development of metastatic ability by cancer cells is a multifactorial process whose temporal events are complex and poorly understood. One step in the metastatic process may involve cell motility. Previous studies reported correlations between motility and metastatic ability. Whether this correlation, seen in cancer cells maintained for long periods of time, is an epiphenomenon developing late in the growth of the cancer as a selection artifact of continuous passage, or is critically required for the acquisition of metastatic ability is unknown. To investigate the relationship between cell motility and the acquisition of metastatic ability, advantage was taken of recently developed DNA transfection methods for inducing high metastatic ability in initially low metastatic cancer cells. The Dunning AT2.1 cell line, a clonal rat prostatic cancer cell line with low metastatic ability, was transfected with a plasmid containing the neomycin resistance gene alone or in combination with the v-Harvey-ras oncogene. A series of the transfected cells was isolated by limiting dilution. After the first in vitro passage following transfection, cells were inoculated into rats to characterize their metastatic ability. The same transfectants were simultaneously studied using our visual grading system of cell motility to study the early motility changes associated with newly acquired metastatic ability. The data demonstrate increased membrane ruffling, pseudopodal extension, and cell translation (translocation) in the v-H-ras-transfected cell lines with high metastatic potential.     

Malignant transformation of human fibroblasts by a transfected N-ras oncogene

The only ras oncogene as yet identified in cells from human fibrosarcomas is N-ras, but the relationship between N-ras oncogene expression and the malignant state of these cell lines is not known. To determine if expression of an N-ras oncogene causes human cells to become malignant, we transfected the N-ras oncogene from human leukemia cell line 8402, cloned into a high expression vector pSV N-ras, into MSU-1.1 cells, a nontumorigenic, infinite life span fibroblast cell strain with a normal morphology and a stable near-diploid karyotype. The transformants formed distinct foci composed of morphologically transformed cells. Cells from such foci expressed higher than normal levels of N-ras protein, exhibited growth factor independence, and formed large colonies in soft agar at a high frequency. Injection of progeny of these focus-derived cells s.c. into athymic mice resulted in progressively growing, invasive malignant tumors (round cell, spindle cell, or giant cell sarcomas) which reached a diameter of 6 mm in 3 to 4 weeks. Injection of focus-derived or tumor-derived cells i.v. resulted in tumors in various organs of the mice. The focus-derived cell strain tested, as well as the majority of the cells derived from the tumor it produced, exhibited the same near-diploid karyotype as the parental MSU-1.1 cells. Cells transfected with an N-ras oncogene that was expressed at a normal level formed only a single, indistinct focus, and cells from that focus were not malignant.     

The transformation of primary and established mouse mammary epithelial cells by p21-ras is concentration dependent

We constructed a retroviral vector (pZSR) which is capable of simultaneously expressing the neomycin resistance gene and the viral ras oncogene. Primary mammary gland epithelial cells were prepared from mid-pregnant mice and infected with this virus. Cell lines with epithelial cell characteristics could be established with a low frequency. High expression of p21 v-ras was observed in these cells. They are tumorigenic and form soft agar colonies dependent on the presence of EGF and insulin in the growth medium but progress to hormone independent growth at higher passage numbers. A cloned cell line of non-tumorigenic, established mammary epithelial cells (NOG8) was also infected with the v-ras expressing virus. Individual cell clones expressing increasing amounts of p21 v-ras were selected. The level of p21 v-ras expression directly influences the morphology of the epithelial cells in culture, determines their cloning efficiency in soft agar and their tumorigenicity.     

The influence of the oncogene N-ras on cell cycle delay in a human melanoma cell line with reduced radioresistance

In a previous study we found that transfection of a human melanoma cell line with the oncogene N-ras led to increased radiosensitivity as measured by clonogenic assays. Since a shift in radiosensitivity is often correlated with altered G2/M delay, we investigated whether this was also the case in this oncogene containing melanoma cell line (IGRras). A human melanoma cell line, stably transfected with mutated N-ras, and its parental cell line transfected with the neomycin phosphotransferase gene only (IGRneo), were irradiated with 5 Gy and cell cycle distribution was measured at hourly time intervals by DNA staining with propidium iodide. Next, the effect of ionising radiation on the duration of the S-phase was determined by pulse labelling cells with BrdUrd before irradiation. Both cell lines showed a radiation induced G2/M delay, which was most prolonged for the ras transfected cell line. After 5 Gy, the S-phase duration was unaltered, although the shape of the relative movement (RM) curves was slightly different. No G1 delay was observed in either cell line. Ras transfection in a melanoma cell line leads to prolonged G2/M delay after radiotherapy. This prolongation is associated with increased radiosensitivity and not with radioresistance. These data throw doubt on the use of oncogene expression or G2/M delay as predictors of radiosensitivity.     

Ha-ras oncogene induction of invasion and metastasis is associated with the activation and redistribution of protease(s) in rat-kidney cells

To investigate the role of oncogenes in the development of metastatic ability, the Ha-ras oncogene was transfected into murine kidney cell line NRK and an alternative murine cell line C127 cells. The resulting transfectants, NRK-Ha and HC127, were assayed for their invasive capability in an in vitro invasion assay and compared with their parental cell lines. All transfected cells were demonstrated to express a 0.6 kb mRNA for the ras oncogene by Northern blot analysis. Using a modified Boyden chamber, we showed that the motile ability of the cell lines, transfected versus non-transfected, were not altered by the introduction of the Ha-ras oncogene. However, ras transfected NRK-Ha cells were able to penetrate and invade through an artificial basement membrane coated filter when compared with the non-transfected parental cell line. Neither the C127 cells nor its ras transfected HC127 cells were able to migrate or penetrate through an artificial basement membrane coated filter. Using immunoblot analyses, we determined that invasive NRK-Ha cells expressed a mature form of protease cathepsin B with a molecular weight between 25-28 kD. Furthermore, using subcellular fractionation and immunoblot, we localized this mature form of cathepsin B to the plasma membrane fraction of the invasive NRK-Ha cells. The plasma membrane fraction of invasive NRK-Ha cells was able to degrade basement membrane components such as fibronectin and the patterns seen were similar to those of purified cathepsin B degradation. Non-invasive NRK, C127 and HC127 cells had only the precursor form of cathepsin B with a molecular weight between 38-45 kD and were unable to degrade basement membrane components. We suggest that the presence and expression of the Ha-ras oncogene alone does not confer metastatic capability, but rather that the ras oncogene must signal specific intracellular redistribution of proteases such as cathepsin B in order to promote tumor cell invasiveness.     

Different malignant phenotypes induced in a stable, subdiploid, benign epithelial clone by DNA transfection.

Progression to malignancy in carcinomas has been studied in a stable, benign, subdiploid, cloned epithelial cell line (A5P/B10) sensitive to Geneticin at 100 micrograms/ml. A total of 28 cell lines were selected for Geneticin - resistance and inoculated into the footpads of syngeneic animals following co-transfection with pSV2neo and genomic DNA, or transfection with plasmid constructs containing neo and the activated Ha-ras oncogene. The behavior of 12 cell lines cotransfected with normal genomic DNA and inoculated into 146 footpads was the same as the A5P/B10 cells. Low grade primary tumors were produced in 122 footpads by 13 cell lines transfected with Ha-ras, and a proportion (61/122) produced well-differentiated lymph node metastases. One of 3 cell lines cotransfected with genomic DNA from a malignant cell line (BC1) produced 8 anaplastic primary tumors with anaplastic metastases. Cell lines from lymph nodes involved by these anaplastic tumors were sensitive to Geneticin, and genomic DNA from 2 clones of these cells failed to produce a malignant phenotype when co-transfected into the A5P/B10 cells. These results indicated that the progression to a malignant phenotype induced in benign cells from a spontaneous epithelial tumor by co-transfection with genomic DNA from malignant cells was different from that induced by the ras oncogene.     

The v-abl, c-fms, or v-myc oncogene induces gamma radiation resistance of hematopoietic progenitor cell line 32d cl 3 at clinical low dose rate

A variety of viral and cellular oncogenes have been described with differing mechanisms of action but with the common property of inducing morphologic alteration of cells in culture. Subclonal lines of oncogene expressing cells have been shown to produce tumors in vivo. Expression of the N-ras oncogene in embryo fibroblast NIH/3T3 cells has been demonstrated to increase radioresistance in vitro, and these results have been confirmed and extended to human cell lines expressing the c-raf oncogene. In the present report, we have examined the effects of expression of the c-fms, v-abl, or v-myc oncogene in a clonal hematopoietic progenitor cell line 32D cl 3. The 32D cell line is nonmalignant in vivo and is dependent upon a source of Interleukin-3 (IL-3) for growth in vitro. The radiation survival of 32D cl 3 cells transfected and expressed in the c-fms oncogene showed significant increase in the radioresistance at both 5 cGy/min and 116 cGy/min. A clone of 32D cl 3 transfected and expressing the v-myc oncogene demonstrated increased radioresistance at both dose rates. Results of split dose experiments suggested significant repair of sublethal irradiation damage of 32D-v-abl cells. Results were compared with expression of the same v-abl oncogene in the NIH/3T3 embryo fibroblast cell line. The data demonstrate that gamma irradiation resistance is significantly increased by each oncogene expressed in 32D cl 3 cells. The data on cell line 32D cl 3 may correlate with the radioresistance of v-abl expressing human hematopoietic cell malignancies treated by irradiation therapy.     

Tumorigenicity of human mesothelial cell line transfected with EJ-ras oncogene

We performed this study to determine whether human mesothelial cells are capable of undergoing neoplastic change in vitro and to observe their interaction with the activated c-Ha-ras (HRAS1) oncogene EJ-ras, which has a role in the development of many malignant human tumors. Mesothelial cells are presumed to be the progenitor cells of malignant mesothelioma, a cancer strongly correlated with asbestos exposure. Previously, we established a non-tumorigenic cell line, MeT-5A, from normal human mesothelial cells after transfection with a plasmid containing the simian virus 40 (SV40) early-region genes. In the present study, we performed transfection of a plasmid containing the EJ-ras gene and the neomycin-resistance gene into these cells and selected a population resistant to G418, a neomycin analogue. Cells from this cell line formed rapidly growing sc tumors in NIH Swiss athymic nude mice, but untransfected with the vector DNA and selected for G418 resistance formed no tumors. The tumors formed by EJ-ras-transfected cells were established in vitro, and cells from these tumor cell lines exhibited a characteristic altered morphology. The cells had the same isoenzyme phenotype as the parent cells, and they expressed the mutant EJ-ras p21 protein. This first demonstration of malignant transformation of human mesothelial cells in vitro may permit molecular analysis of mesothelial carcinogenesis.     

Growth in culture and tumorigenicity after transfection with the ras oncogene of liver epithelial cells from carcinogen-treated rats

Two epithelial cell lines designated LE/2 and LE/6 were established from cells isolated by centrifugal elutriation from the livers of carcinogen-treated rats. Both cell lines exhibit some characteristics of fetal liver cells, such as the expression of the 2.3-kilobase alpha-fetoprotein mRNA, aldolase A, and lactate dehydrogenases 4 and 5. Primary cultures contain gamma-glutamyl transferase-positive cells which do not proliferate in vitro. After the first passage, the LE/2 and LE/6 cell lines are uniformly gamma-glutamyl transferase negative. Neither cell line is transformed as assayed by morphology, anchorage-independent growth, or tumor formation in nude mice. By the 50th passage, LE/6 cells form numerous colonies in soft agar in the presence of epidermal growth factor, while no colonies grow in medium lacking this growth factor. Clonal cell populations derived from five epidermal growth factor-induced soft agar colonies were not tumorigenic in nude mice. This indicates that, although epidermal growth factor-responsive late passage cells had acquired some of the phenotypic properties commonly associated with tumor cells, these cells were not fully transformed. Transformation of LE/6 cells was accomplished by transfection of the rasH oncogene (EJ). Subcutaneous inoculation of rasH (EJ)-transfected LE/6 cells produced tumors at the site of injection with histological features of moderate to well-differentiated trabecular hepatocellular carcinomas. Tumor cell lines derived from the nude mouse tumors are gamma-glutamyl transferase positive and express alpha-fetoprotein mRNA. One clonal cell line expresses both alpha-fetoprotein and albumin mRNA. These results show that nonparenchymal liver epithelial cells transfected with an activated oncogene can give rise to differentiated hepatocellular tumors similar to those induced in livers of rats fed a carcinogenic diet.     

Constitutive expression of the c-H-ras oncogene inhibits doxorubicin-induced apoptosis and promotes cell survival in a rhabdomyosarcoma cell line.

Drugs used in anti-cancer chemotherapy are thought to exert their cytotoxic action by induction of apoptosis. Genes have been identified which can mediate or modulate this drug-induced apoptosis, among which are c-myc, p53 and bcl-2. Since expression of oncogenic ras genes is a frequent observation in human cancer, we investigated the effects of the c-H-ras oncogene on anti-cancer drug-induced apoptosis. Apoptosis induced by a 2 h doxorubicin exposure was measured by in situ nick translation and flow cytometry in a rat cell line (R2T24) stably transfected with the c-H-ras oncogene and in a control cell line (R2NEO) transfected only with the antibiotic resistance gene neo. Both cell lines (R2T24 and R2NEO) had nearly identical growth characteristics, including cell doubling time, distribution over the cell cycle phases and plating efficiency in soft agar. Doxorubicin exposure of the R2NEO cells led to massive induction of apoptosis. In contrast, R2T24 cells, expressing the c-H-ras oncogene, showed significantly less apoptosis after doxorubicin incubation. Doxorubicin induced approximately 3- to 5-fold less cytotoxicity in the R2T24 cells than in the R2NEO cells, as determined by clonogenic assay in soft agar. No difference was observed in intracellular doxorubicin accumulation between the two cell lines, indicating that the classical, P-glycoprotein-mediated multidrug resistance phenotype is not involved in the observed differences in drug sensitivity. In conclusion, our data show that constitutive expression of the c-H-ras oncogene suppresses doxorubicin-induced apoptosis and promotes cell survival, suggesting that human tumours with ras oncogene expression might be less susceptible to doxorubicin treatment.     

Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras

We sought to determine whether the transfection of tumorigenic but not metastatic cells with the activated c-Ha-ras oncogene was invariably associated with acquisition of the metastatic phenotype. Three clonally derived lines of the K-1735 murine melanoma, characterized as nonmetastatic or poorly metastatic, were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, that confers resistance to neomycin (pSV2neoEJ). Cells transfected with pSV2neo, a plasmid containing the neo gene, served as controls for the procedure of Polybrene-mediated transfection. All cell lines were injected into syngeneic C3H/HeN and into athymic mice, and the results were compared with those produced by highly metastatic K-1735 M-2 cells. Although the pSV2neoEJ-transfected cells produced more rapidly growing s.c. tumors than the control cell lines did, the incidence of spontaneous metastasis was not increased. Following i.v. inoculation, the c-Ha-ras transfectants were retained in lung vasculature in greater proportions than pSV2neo counterpart transfectants were. The c-Ha-ras transfectants also produced significantly more lung tumor colonies, which grew faster than the few lung tumor colonies in mice given injections of control melanoma cells. We concluded that transfection of the activated c-Ha-ras oncogene into nonmetastatic K-1735 melanoma cells leads to accelerated tumor growth in vivo and can confer the ability to form lung colonies after i.v. injection but not the ability to metastasize from a primary s.c. tumor.     

Transformation of normal homologous cells by a spontaneously activated Ha-ras oncogene

Several tumor-derived oncogenes have been shown to independently act as complete carcinogens following transfection into target cells from established tissue culture lines. However, the number and types of oncogenes required to transform primary cultures of normal mammalian cells is unclear. To clarify this issue in a simplified model system, we transfected genomic DNA from a naturally occurring rat tumor into NIH/3T3 cells as well as into early passage rat embryo fibroblasts. The 3T3 cells were transformed with high efficiency to malignant phenotypes; the rat embryo cells were transformed at lower frequencies following cotransfection with a selectable neomycin resistance marker and treatment with Geneticin (G418). The transformed rat cells had cancerous phenotypes as determined by in vitro, cytogenetic, and in vivo criteria. Moreover, the transformed mouse and rat cells contained new tumor DNA-derived nucleotide sequences homologous to the activated human Ha-ras oncogene. Elevated levels of Ha-ras-specific mRNA, as well as enhanced expression of the Mr 21,000 oncogene product, were detected in the transformed cells. Therefore, under well-defined experimental conditions, a spontaneously activated Ha-ras oncogene from a naturally occurring tumor was able to independently transform normal, homologous cells to a malignant phenotype.     

Beta catenin is associated with breast cancer progression in vitro

Cancer is induced by a series of genetic alterations that lead to changes in the normal mechanisms controlling cell proliferation, differentiation, cell death, or genomic instability. The MCF-10F, a spontaneously immortalized human breast epithelial cell line, treated with benzo(a)pyrene and then transfected with the c-Ha-ras oncogene was used in these studies. The aim was to define the phenotypic alterations associated with the carcinogenic process. Carcinogen-treated and c-Ha-ras-transfected cells showed a progression of changes in the morphology as seen by transmission electron microscopy, anchorage-independent growth, invasiveness and capability of tumor formation in the SCID mice, as well as altered oncoprotein expression. Furthermore, these cells showed an increased expression of MDM2, Int-2 (FGF-3) and beta catenin in comparison to control MCF-10F as determined by fluorescence staining coupled with confocal microscopy. The MDM2, Int-2 (FGF-3) expressions were increased in cell lines transfected with the c-Ha-ras with or without carcinogen treatment as well as the tumor cell line derived from a tumor formed in the SCID mice in comparison to control cell line MCF-10F. However, beta catenin was only increased in the most aggressive tumorigenic cell lines in comparison with MCF-10F cell line and non-transfected ones. It can be concluded that malignant progression is a stepwise process and tumor growth occurs after a series of molecular events that parallel morphological changes indicative of cell transformation.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Cell surface glycosylation changes accompanying immortalization and transformation of normal human mammary epithelial cells

Fluorescent lectin binding to cell surfaces was quantitatively analysed by flow cytometry on mortal human breast epithelial cells MCF-10M, the immortalized cell line MCF-10A derived from MCF-10M and sublines of MCF-10A transfected with the neomycin resistance gene (MCF-10Aneo), the c-Ha-ras protooncogene (MCF-10AneoN), or transfected and transformed with the c-Ha-ras activated oncogene (MCF-10AneoT). Immortal MCF-10A cells bound 10-fold more peanut agglutinin (PNA) and soy bean agglutinin (SBA) than did MCF-10M cells. Transformed MCF-10AneoT cells bound approximately ten times more PNA than did non-transformed cells transfected with protooncogene (MCF-10AneoN). Treatment of the transfectants with neuraminidase abrogated the differences in PNA-binding and reduced the differences in SBA binding. SDS-PAGE separation of PNA binding glycoproteins revealed different patterns for all MCF-10A derived sublines.