Ectopic expression of MITF, a gene for Waardenburg syndrome type 2, converts fibroblasts to cells with melanocyte characteristics.

MITF (microphthalmia-associated transcription factor) encodes a transcription factor with a basic-helix-loop-helix-zipper (bHLH-Zip) motif. MITF mutations occur in patients with Waardenburg syndrome type 2, a disorder associated with melanocyte abnormalities. Here we show that ectopic expression of MITF converts NIH/3T3 fibroblasts into cells with characteristics of melanocytes. MITF transfectants formed foci of morphologically altered cells, which resemble those induced by oncogenes, but did not exhibit malignant phenotypes. Instead, they contained dendritic cells that express melanogenic marker proteins such as tyrosinase and tyrosinase-related protein 1. Most cloned cells of MITF transfectants exhibited dendritic morphology and expressed melanogenic markers, but such properties were not observed in cells transfected with closely related TFE3 cDNA. Our findings indicate that MITF is critically involved in melanocyte differentiation.

Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Viral genes involved in leukemogenesis. I. Generation of recombinants between oncogenic and nononcogenic mouse type-C viruses in tissue culture.

An approach toward elucidation of the mechanisms of action of mammalian leukemia viruses has been made by the generation in tissue culture of recombinant viruses between a potent murine leukemia virus (MuLV), Rauscher-MuLV, and an endogenous xenotropic mouse type-C virus, BALB:virus-2, without known malignant potential. Using a double selection system devised to select against the temperature-sensitive (ts) lesion associated with a mutant of Rauscher-MuLV and the xenotropic host range of BALB:virus-2, recombinant viruses were obtained at frequencies ranging from 0.01 to 0.1%. Recombinant viruses were identified on the basis of the type specific antigenic determinants in the translational products of gag (p15, p12, p30, and p10 proteins), pol (reverse transcriptase), and env (gp70 glycoprotein) genes. By this approach, the partial genetic maps of a large number of recombinants were obtained. The fact that p10 of Rauscher-MuLV ts 25, the mutant utilized, was the only protein uniformly lacking in recombinant viruses, localized the lesion inhibiting gag precursor cleavage in this mutant at the carboxy terminus of its gag gene. The recombinant viruses demonstrated two host range phenotypes as defined by Fv-1 host cell restriction. In each case, NB-tropic recombinants possessed the p30 of BALB:virus-2 p30. Thus, it was possible to assign the site of Fv-1 action at, or closely linked, to the viral p30. The target within the viral genome of a second host restriction was also mapped. A serum factor, previously shown to specifically inactivate xenotropic virus infectivity, was demonstrated to exert its action on the viral env gene product. The system described here allows the generation of specific recombinant genotypes that should be useful in defining those regions of the viral genome involved in leukemogenesis.

Antigenic properties of murine sarcoma virus-transformed BALB-3T3 nonproducer cells.

The isolation of clonal lines of murine sarcoma virus-transformed, non-producer BALB/3T3 cells has provided a model system for determining whether RNA tumor virus-transformed cells possess virus-specific transplantation antigens. MSV nonproducer cells (K-234) were clonally derived from an inbred mouse cell line, BALB/3T3. A parallel virus-producing cell line was obtained by infection of the MSV nonproducer cells with Rauscher leukemia virus. K-234 was much more tumorigenic than K-234(R). Preimmunization of syngeneic mice with either K-234(R) or with UV-inactivated Rauscher leukemia virus induced transplantation resistance to subsequent challenge with K-234(R), but not with K-234. In contrast, mice preimmunized with nonproducer cells were not made resistant to subsequent challenge with the homologous cells. Antisera prepared from mice immunized with K-234(R) were specifically cytotoxic and positive by fluorescent antibody staining for K-234(R) target cells, but not to either BALB/3T3 or K-234. The results show that MSV nonproducer cells lack detectable transplantation antigens and suggest that the transplantation resistance to the producing cells is attributable to maturing virus at the cell surface.

Genetic factors influencing C-type RNA virus induction.

Genetic factors were studied which affect the inducibility of C-type RNA viruses from embyro cultures of mouse strains with high and low incidence of spontaneous leukemia. Virus was inducible by chemical treatment from secondary embryo cultures of several inbred strains. Both virus inducibility and the capacity of the virus to persist in cells from which it was activated were found to be inherited as dominant genetic characteristics. The results show that the factors controlling virus activation and persistence in culture also play an important role in spontaneous virus expression in the animal.

BALB- and Harvey-murine sarcoma virus transformation of a novel lymphoid progenitor cell.

BALB- and Harvey-murine sarcoma viruses (MSV) comprise a family of retroviruses whose mouse- and rat-derived onc genes are closely related. These viruses induce sarcomas and erythroleukemias in susceptible animals. An in vitro colony assay that detects transformation of lymphoid cells by Abelson-murine leukemia virus was used to demonstrate that BALB- and Harvey-MSV transform a novel hematopoietic cell both in culture and in vivo. Bone marrow colony formation was sarcoma virus dependent, followed single-hit kinetics, and required the presence of mercaptoethanol in the agar medium. BALB- and Harvey-MSV-induced colonies could be established in culture as continuous cell lines that demonstrated unrestricted self-renewal capacity and leukemogenicity in vivo. The cells had a blast cell morphology and lacked detectable markers of mature cells within the myeloid or erythroid series. They also lacked detectable immunoglobulin mu chain or Thy-1 antigen, markers normally associated with committed cells of the B and T lymphoid lineages, respectively. However, the transformants contained very high levels of terminal deoxynucleotidyl transferase (TdT), an enzyme believed to be specific to early stages within the lymphoid differentiation pathway. This phenotype distinguishes these BALB- and Harvey-MSV transformants from any previously reported hematopoietic targets of transforming retroviruses, including the pre-B lymphoid cell transformed by Abelson-MuLV under identical assay conditions. These newly identified lymphoid progenitor cell transformants may provide an important means of studying early stages of lymphoid ontogeny and the possible role of TdT in lymphoid development.

Wild mouse RNA tumor viruses. A nongenetically transmitted virus group closely related to exogenous leukemia viruses of laboratory mouse strains.

Type-C RNA viruses isolated from wild mice are causative of naturally occurring neoplasia and neurologic diseases. Biochemical and immunologic characterization of this virus group revealed that amphotropic viruses isolated from wild mice trapped in separate geographical areas are indistinguishable, whereas amphotropic and ecotropic viruses naturally infecting the same animal are env gene variants. Molecular hybridization studies established that neither host range variant is endogenous to the Mus musculus genome, although each demonstrates partial nucleotide sequence homology. Wild mouse type-C viruses exhibited much closer molecular and antigenic relatedness to the exogenous virus subgroup (Friend-, Moloney-, and Rauscher-MuLV) than to prototype endogenous viruses isolated from laboratory mouse strains. The evidence indicates that exogenous mouse type-C viruses have been maintained in nature over a long period of evolution as a separate virus group, causative of tumors in mice by a mechanism solely involving their transmission as infectious agents.

Isolation and characterization of C-type viral gene products of virus-negative mouse cells.

Antigens which immunologically cross-react with two mouse C-type viral polypeptides, p30 and p12, are present at very low levels in normal virus-negative mouse cells. These two antigens have been purified by 50-300-fold from cell extracts and shown to cochromatograph with the corresponding labeled viral polypeptides in several systems. Their type-specific antigenicities are shown to be distinct from those of previously tested MuLV isolates suggesting that they may be components of a new class of endogenous C-type virus. The methods utilized in the present studies for concentration of virus-specific antigens of normal mouse cells provide an approach for detection of C-type viral antigens in cells of other species.

Modulation of keratinocyte growth factor and its receptor in reepithelializing human skin.

We investigated the expression and distribution of keratinocyte growth factor (KGF) (FGF-7) and its receptor (KGFR) during reepithelialization of human skin. KGF mRNA levels increased rapidly by 8-10-fold and remained elevated for several days. In contrast, KGFR transcript levels decreased early but were significantly elevated by 8-9 d. A KGF-immunoglobulin G fusion protein (KGF-HFc), which specifically and sensitively detects the KGFR, localized the receptor to differentiating keratinocytes of control epidermis, but revealed a striking decrease in receptor protein expression during the intermediate period of reepithelization. Suramin, which blocked KGF binding and stripped already bound KGF from its receptor, failed to unmask KGFRs in tissue sections from the intermediate phase of wound repair. The absence of KGFR protein despite increased KGFR transcript levels implies functional receptor downregulation in the presence of increased KGF. This temporal modulation of KGF and KGFRs provides strong evidence for the functional involvement of KGF in human skin reepithelialization.

Comparative analysis of p73 and p53 regulation and effector functions.

p53 is mutated in approximately 50% of human cancers, whereas mutations of the related p73 gene are rare. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. We show that p73 isoforms, p73alpha and p73beta, can each induce permanent growth arrest with markers of replicative senescence when overexpressed in a tetracycline-regulatable manner in human cancer cells lacking functional p53. Human homologue of mouse double minute 2 gene product (hMDM2), but not an NH(2)-terminal deletion mutant, coimmunoprecipitated with p73alpha or p73beta, and inhibited p73 transcriptional activity as with p53. In contrast to p53, ectopically expressed hemagglutinin (HA)-tagged p73 proteins were not stabilized by treatment with several DNA damaging agents. Furthermore, unlike normal p53, which increases in response to DNA damage due to enhanced protein stability in MCF7 cells, endogenous p73 protein levels were not increased in these cells under the same conditions. Thus, although p73 has an ability, comparable to that of p53, to suppress tumor cell growth in p53-deficient cells, p73 induction is regulated differently from p53. These findings suggest that the selective pressures for p53 rather than p73 inactivation in tumors may reflect their differential responses to stresses such as DNA damage, rather than their capacities to induce permanent growth arrest or apoptosis programs.

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

Progesterone-dependent expression of keratinocyte growth factor mRNA in stromal cells of the primate endometrium: keratinocyte growth factor as a progestomedin.

In vitro studies have shown that keratinocyte growth factor (KGF, also known as FGF-7) is secreted by fibroblasts and is mitogenic specifically for epithelial cells. Therefore, KGF may be an important paracrine mediator of epithelial cell proliferation in vivo. Because stromal cells are thought to influence glandular proliferation in the primate endometrium, we investigated the hormonal regulation and cellular localization of KGF mRNA expression in the rhesus monkey uterus. Tissues were obtained both from naturally cycling monkeys in the follicular and luteal phases of the cycle, and from spayed monkeys that were either untreated or treated with estradiol (E2) alone, E2 followed by progesterone (P), E2 plus P, or E2 plus P plus an antiprogestin (RU 486). Northern blot analysis of total RNA with 32P-labeled probes revealed that the level of KGF mRNA in the endometrium was 70-100-fold greater in the luteal phase or after P treatment than in untreated, E2-treated, or follicular phase animals. Northern analysis also showed that KGF mRNA was present in the myometrium but was unaffected by hormonal state. RU 486 treatment prevented the P-induced elevation of endometrial KGF mRNA. P-dependent elevation of endometrial KGF expression was confirmed by measurement of KGF protein in tissue extracts using a two-site enzyme-linked immunosorbent assay. In situ hybridization with nonradioactive digoxigenin-labeled cDNA probes revealed that the KGF mRNA signal, which was present only in stromal and smooth muscle cells, was substantially increased by P primarily in the stromal cells located in the basalis region. Smooth muscle cells in the myometrium and the walls of the spiral arteries also expressed KGF mRNA, but the degree of this expression did not differ with hormonal state. P treatment led to increased proliferation in the glandular epithelium of the basalis region and to extensive growth of the spiral arteries. We conclude that the P-dependent increase in endometrial KGF resulted from a dual action of P: (a) a P-dependent induction of KGF expression in stromal cells, especially those in the basalis (zones III and IV), and (b) a P-dependent increase in the number of KGF-positive vascular smooth muscle cells caused by the proliferation of the spiral arteries. KGF is one of the first examples in primates of a P-induced, stromally derived growth factor that might function as a progestomedin.

Specific receptor detection by a functional keratinocyte growth factor-immunoglobulin chimera.

Fibroblast growth factor receptors (FGFRs) are encoded by at least four distinct highly conserved genes, and alternative splicing generates multiple gene products. The close relationship among different FGFRs has greatly increased the difficulty in generating specific immunochemical probes. As an alternative strategy, we constructed a fusion protein comprising keratinocyte growth factor (KGF) and an IgG1 Fc domain (HFc). The chimeric molecule was efficiently secreted from transfectants as a disulfide-linked dimer that bound KGFRs with high affinity. Moreover, the KGF-HFc, like native KGF, induced DNA synthesis by epithelial cells implying normal functional receptor activation. Because it retained the convenient detection properties of an immunoglobulin, it was possible to use the KGF-HFc in ligand-mediated histochemical analysis of KGFRs. Flow cytometry revealed KGF-HFc chimera detection of the KGFR, an alternative FGFR2 product, but not FGFR1 (flg) or FGFR2 (bek). Histochemical analysis of normal skin demonstrated the specific localization of KGFRs within the spinous layer, a zone of epithelial cell differentiation. KGFRs were also localized to epithelial cells within a specific region of the hair follicle, and they were not detectable in cells of the sweat gland. Tissue sections of soft palate and tonsil, two examples of nonkeratinizing epithelium, revealed staining of stratum spinosum and some staining of the basal cell layer as well. Neither salivary gland epithelium nor lymphoid cells were positive. The ciliated epithelium of the trachea exhibited KGFR expression in intermediate and basal cell layers. In striking contrast to the normal pattern of staining in the adjacent epithelium, a squamous cell carcinoma of skin lacked detectable KGFRs. Our present findings suggest that growth factor-Ig fusion proteins may be generally applicable in ligand-mediated histochemical detection and localization of growth factor receptors.

Growth factors and cancer.

Signaling pathways that mediate the normal functions of growth factors are commonly subverted in cancer. Oncogenes identified by a variety of approaches have been shown to function at critical steps in mitogenic signaling. Progression through the cell cycle requires the coordinated actions of members of two complementary classes of growth factors, and oncogenes appear to replace the actions of one set of these growth factors. Growth factors can also influence normal cell differentiation, and constitutive activation of growth-promoting pathways in cancer cells can modulate the cell phenotype as well. Paracrine actions of growth factors and cytokines may also influence the stepwise series of genetic events that lead to malignancy. New approaches for cancer therapy are being developed that intervene at various steps in growth factor signaling pathways.

Frequent activation of c-kis as a transforming gene in fibrosarcomas induced by methylcholanthrene.

The DNA's from two of four methylcholanthrene-induced mouse fibrosarcomas contained transforming genes that were identical in their pattern of restriction endonuclease resistance to inactivation of biologic activity. This transforming gene was identified as the activated homolog of the Kirsten murine sarcoma virus onc gene, v-kis. The finding that a defined carcinogen reproducibly leads to activation of kis as a transforming gene should be of value in elucidating the role of oncogenes in the neoplastic process.

High-frequency C-type virus induction by inhibitors of protein synthesis.

When inhibitors of protein synthesis are added to BALB/c mouse cells in culture, induction of naturally integrated C-type RNA virus occurs in a high percentage of cells. The action of protein synthesis inhibitors differs from that of halogenated pyrimidines, another class of virus inducers, in their effects on biologically distinguishable viruses. The use of such inhibitors to study integrated virus expression provides a means for studying gene regulation in mammalian cells.

Segregation of loci for C-type virus induction in strains of mice with high and low incidence of leukemia.

Multiple genetic loci for induction of murine leukemia viruses are demonstrated in cells of the high leukemic incidence C58 mouse strain. The biologic properties of viruses at C58 inducibility loci are clearly distinguishable from those of viruses activated from mouse cells containing a locus for virus induction of the low leukemia incidence BALB/c strain. These findings are consistent with the hypothesis that the genes for virus induction in normal mouse embryo cells represent viral structural information.

Basis for the acquisition of malignant potential by mouse cells cultivated in vitro.

Balb/c mouse embryo lines maintained in culture for over 200 generations under conditions that minimize cell-cell contact do not become tumorigenic. Lines cultivated under conditions where there is extensive cell contact become tumor-producing within 30 generations. The tissue-culture property that correlates best with tumorigenicity is the loss of contact inhibition of cell division.

Continuous lines of basophil/mast cells derived from normal mouse bone marrow.

Nonadherent tissue culture cell lines were established from normal bone marrow of a variety of mouse strains. The lines possessed morphological and histochemical markers of the basophil/mast cell and contained committed stem cells for metachromatic cells. Their derivation from normal marrow and their lack of tumorigenicity despite long-term culture makes these cell lines potentially important for studies of the mechanisms of allergic reactions and inflammation as well as the differentiation pathways involving this subset of hematopoietic cells.

Neoplastic conversion of human keratinocytes by adenovirus 12-SV40 virus and chemical carcinogens.

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12 and simian virus 40 (Ad 12-SV40) were transformed by treatment with the chemical carcinogens N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes.