Independent molecular pathways in initiation and loss of hormone responsiveness of breast carcinomas

These studies were set up to determine whether those oncogenes participating in the initiation of mammary carcinogenesis (for example, ras oncogenes) play a direct role in the outcome of events associated with the late stages of tumor development such as loss of hormone dependency. Mammary carcinomas induced by a single carcinogenic insult in pubescent rats was selected as an in vivo model system with direct relevance to human breast cancer. Acquisition of hormone-independent growth in these carcinogen-induced tumors was found to be independent of the activation of ras oncogenes during the early stages of carcinogenesis. In agreement with these observations, introduction of a human ras oncogene into human MCF-7 breast carcinoma cells did not abrogate their hormonal dependency for growth in vivo. These findings suggest that those events responsible for the critical stages of breast cancer development occur independently and in an uncoordinated manner.     

Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes.

Mammalian 3T3-L1 cells differentiate into adipocytes after continuous exposure to pharmacological doses of insulin or physiological doses of insulin-like growth factor I (IGF-1). Expression of transfected ras oncogenes led to differentiation of these cells into adipocytes in the absence of externally added insulin or IGF-I. Cells transfected with normal ras genes or the tyrosine kinase trk oncogene did not differentiate. Transfection with a dominant inhibitory ras mutant resulted in inhibition of differentiation. Exposure of untransfected 3T3-L1 cells to insulin stimulated formation of the active Ras.GTP complex. These observations indicate that Ras proteins participate in signal transduction pathways initiated by insulin and IGF-I in these cells.     

Activation of ras oncogenes preceding the onset of neoplasia

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.     

Transformation by oncogenes encoding protein kinases induces the metastatic phenotype

Oncogenes encoding serine/threonine or tyrosine kinases were introduced into the established rodent fibroblast cell line NIH 3T3 and tested for tumorigenic and metastatic behavior in T cell-deficient nude mice. Transforming oncogenes of the ras family were capable of converting fibroblast cell lines to fully metastatic tumors. Cell lines transformed by the kinase oncogenes mos, raf, src, fes, and fms formed experimental metastases and (in some cases) these genes were more efficient at metastatic conversion than a mutant ras gene. In contrast, cells transformed by either of two nuclear oncogenes, myc or p53, were tumorigenic when injected subcutaneously but were virtually nonmetastatic after intravenous injection. These data demonstrate that, in addition to ras, a structurally divergent group of kinase oncogenes can induce the metastatic phenotype.     

Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain

A cytoplasmic protein that greatly enhances the guanosine triphosphatase (GTPase) activity of N-ras protein but does not affect the activity of oncogenic ras mutants has been recently described. This protein (GAP) is shown here to be ubiquitous in higher eukaryotes and to interact with H-ras as well as with N-ras proteins. To identify the region of ras p21 with which GAP interacts, 21 H-ras mutant proteins were purified and tested for their ability to undergo stimulation of GTPase activity by GAP. Mutations in nonessential regions of H-ras p21 as well as mutations in its carboxyl-terminal domain (residues 165-185) and purine binding region (residues 117 and 119) did not decrease the ability of the protein to respond to GAP. In addition, an antibody against the carboxyl-terminal domain did not block GAP activity, supporting the conclusion that GAP does not interact with this region. Transforming mutations at positions 12, 59, and 61 (the phosphoryl binding region) abolished GTPase stimulation by GAP. Point mutations in the putative effector region of ras p21 (amino acids 35, 36, and 38) were also insensitive to GAP. However, a point mutation at position 39, shown previously not to impair effector function, did not alter GAP-p21 interaction. These results indicate that GAP interaction may be essential for ras p21 biological activity and that it may be a ras effector protein.     

Elevated levels of glucose transport and transporter messenger RNA are induced by ras or src oncogenes

An accelerated rate of glucose transport is among the most characteristic biochemical markers of cellular transformation. To study the molecular mechanism by which transporter activity is altered, cultured rodent fibroblasts transfected with activated myc, ras, or src oncogenes were used. In myc-transfected cells, the rate of 2-deoxy-D-glucose uptake was unchanged. However, in cells transfected with activated ras and src oncogenes, the rate of glucose uptake was markedly increased. The increased transport rate in ras- and src-transfected cells was paralleled by a marked increase in the amount of glucose transporter protein, as assessed by immunoblots, as well as by a markedly increased abundance of glucose transporter messenger RNA. Exposure of control cells to the tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) for 18 hours had a similar effect of increasing the rate of glucose transport and the abundance of transporter messenger RNA. For ras, src, and TPA, the predominant mechanism responsible for activation of the transport system is increased expression of the structural gene encoding the glucose transport protein.     

Primary rat embryo cells transformed by one or two oncogenes show different metastatic potentials

Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.     

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.     

Isolation, characterization, and chromosome assignment of mouse N-ras gene from carcinogen-induced thymic lymphoma

Treatment of mice with the carcinogen N-methylnitrosourea results in the development of thymic lymphomas with frequent involvement of the N-ras oncogene. The activated mouse N-ras gene was isolated from one of these lymphomas and, by transformation in concert with restriction digestion, a map of the gene was prepared and its approximate boundaries were determined. By means of somatic cell hybrids the normal N-ras gene was found to be unlinked to other members of the ras gene family.     

The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation

Identification of genes that function to protect cells from radiation damage is an essential step in understanding the molecular mechanisms by which mammalian cells cope with ionizing radiation. The intrinsic radiation resistance (D0) of NIH 3T3 cells was markedly and significantly increased by transformation with ras oncogenes activated by missense mutations. This radiobiologic activity appeared to be a specific consequence of the ras mutations rather than of transformation, since revertant cells that contained functional ras genes (but were no longer phenotypically transformed) retained their increased D0's.     

Site-specific integration of H-ras in transformed rat embryo cells

A karyotypic analysis was performed on seven independently derived clones of primary rat embryo cells transformed by the ras oncogene plus the cooperating oncogene myc. The transfected oncogenes were sometimes present in amplified copy number, with heterogeneity in the levels of amplification. Some chromosomal features, such as aberrantly banding regions and double-minute chromosomes, typical of cells carrying amplified genes, were also seen in three of the seven cell lines. Underlying this heterogeneity there was an unexpected finding. All seven lines showed a common integration site for ras on the q arm of rat chromosome 3 (3q12), though some lines also had other sites of integration. In four of the lines integration of ras was accompanied by deletion of the p arm of chromosome 3 or its possible translocation to chromosome 12.     

Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk assessment

The validity of mouse liver tumor end points in assessing the potential hazards of chemical exposure to humans is a controversial but important issue, since liver neoplasia in mice is the most frequent tumor target tissue end point in 2-year carcinogenicity studies. The ability to distinguish between promotion of background tumors versus a genotoxic mechanism of tumor initiation by chemical treatment would aid in the interpretation of rodent carcinogenesis data. Activated oncogenes in chemically induced and spontaneously occurring mouse liver tumors were examined and compared as one approach to determine the mechanism by which chemical treatment caused an increased incidence of mouse liver tumors. Data suggest that furan and furfural caused an increased incidence in mouse liver tumors at least in part by induction of novel weakly activating point mutations in ras genes even though both chemicals did not induce mutations in Salmonella assays. In addition to ras oncogenes, two activated raf genes and four non-ras transforming genes were detected. The B6C3F1 mouse liver may thus provide a sensitive assay system to detect various classes of proto-oncogenes that are susceptible to activation by carcinogenic insult. As illustrated with mouse liver tumors, analysis of activated oncogenes in spontaneously occurring and chemically induced rodent tumors will provide information at a molecular level to aid in the use of rodent carcinogenesis data for risk assessment.     

A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants

The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.     

Protein-specific helper T-lymphocyte formation initiated by dendritic cells

Antibody responses to hapten-polypeptide conjugates require peptide-specific helper T cells. The latter can be primed in tissue culture by providing small numbers of dendritic cells. Primed, irradiated helper T cells then induce B-cell growth and differentiation in the apparent absence of dendritic cells. Both stages of the antibody response--the induction of helper T lymphoblasts by dendritic cells and the delivery of help from T to B cell--occur in discrete cell aggregates that can be isolated by velocity sedimentation. If helper T blasts revert to smaller "memory" lymphocytes, dendritic cells again are needed to initiate the antibody response.     

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.     

Tripeptide structure of bursin, a selective B-cell-differentiating hormone of the bursa of fabricius

Differentiation of lymphoid precursor cells in a variety of species is induced by polypeptide hormones such as thymopoietin for T cells and bursin for B cells. In the present experiments, bursin isolated from the bursa of Fabricius of chicken was found to induce the phenotypic differentiation of mammalian and avian B precursor cells but not of T precursor cells in vitro. Similarly, bursin increased cyclic guanosine monophosphate in cells of the human B-cell line Daudi but not in cells of the human T-cell line CEM. These inducing properties of bursin are the reverse of the inducing properties of thymopoietin produced by the thymus and are appropriate to a physiological B-cell-inducing hormone. A tripeptide sequence (lysyl-histidyl-glycyl-amide) was determined for bursin and confirmed by synthesizing this proposed structure and demonstrating chemical identity of the natural and synthetic peptides. Similarity of biological action was indicated in induction assays by elevation of cyclic adenosine monophosphate and guanosine monophosphate in Daudi B cells but not in CEM T cells.     

Transfection of v-rasH DNA into MCF-7 human breast cancer cells bypasses dependence on estrogen for tumorigenicity

The natural history of estrogen-responsive breast cancers often involves a phenotypic change to an estrogen-unresponsive, more aggressive tumor. The human breast cancer cell line, MCF-7, which requires estradiol for tumor formation in vivo and shows growth stimulation in response to estradiol in vitro, is a model for hormone-responsive tumors. The v-rasH onc gene was transfected into MCF-7 cells. The cloned MCF-7ras transfectants, which expressed the v-rasH messenger RNA and v-rasH p21 protein (21,000 daltons), were characterized. In contrast to the parental cell line, MCF-7ras cells no longer responded to exogenous estrogen in culture and their growth was minimally inhibited by exogenous antiestrogens. When tested in the nude mouse, the MCF-7ras cells were fully tumorigenic in the absence of estrogen supplementation. Thus, cells acquiring an activated onc gene can bypass the hormonal regulatory signals that trigger the neoplastic growth of a human breast cancer cell line.     

Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins

Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.     

Inhibition of purine nucleoside phosphorylase by 8-aminoguanosine: selective toxicity for T lymphoblasts

The guanosine analog 8-aminoguanosine is an effective inhibitor of the purine degradative enzyme purine nucleoside phosphorylase, both in vitro and in intact lymphoid cells. In a human lymphoblast tissue culture system, 8-aminoguanosine, in combination with low concentrations of 2'-deoxyguanosine, causes toxicity toward T cells but not B cells. The selective T cell toxicity correlates with increased accumulation of deoxyguanosine triphosphate in the treated T lymphoblasts.