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S A Aaronson

Publications and source records attributed to S A Aaronson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas.

In the present study we demonstrate that erbB-3 and erbB-2 cooperate in neoplastic transformation. Under conditions in which neither gene alone induced transformation, they readily transformed NIH3T3 cells if co-expressed. Furthermore, at high expression levels of ErbB2 which cause transformation, ErbB3 enhanced focus formation by one order of magnitude. Synergy required an intact ErbB2 extracellular domain and tyrosine kinase activity. Cooperation between ErbB3 and ErbB2 involved heterodimerization and increased tyrosine phosphorylation of ErbB3. Signaling by the heterodimer resulted in increased PI 3-kinase recruitment as well as quantitative and qualitative differences in substrate phosphorylation. Evidence for signaling by an active ErbB3-ErbB2 heterodimer in four mammary tumor cell lines indicated relevance of this mechanism for human neoplasia. Our detection of the NDF/heregulin transcript in NIH3T3 cells implicates an autocrine loop involving this ligand in signaling by the ErbB3-ErbB2 heterodimer in the model system, whereas heregulin-independent mechanisms likely exist for cooperative signaling by ErbB3 and ErbB2 chronically activated in some human mammary carcinomas.

3T3 Cells↗
PubMed1995

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.

3T3 Cells↗
PubMed1994

A novel dual specificity phosphatase induced by serum stimulation and heat shock.

To identify new members of a family of protein-tyrosine phosphatases (PTPs), of which VH1 is prototype, we screened a B5/589 human mammary epithelial cell cDNA library by low stringency hybridization with probes for the catalytic domains of the human VHR and mouse 3CH134 phosphatases. Two overlapping clones of 1.8 and 2.5 kilobase pairs were detected by 3CH134 but not VHR probes. Sequence analysis of the largest clone, B23, revealed a 2470-nucleotide open reading frame encoding a novel protein. Within the 397 amino acid sequence, the HCXAGXXR signature sequence for PTPs was located at positions 261-268. The closest similarities were to 3CH134, its human homolog CL100, and PAC-1, PTPs induced as early response genes to mitogen stimulation. Less relatedness was observed with VHR and VH1 dual specificity phosphatases of human and vaccinia virus, respectively. A bacterially expressed recombinant protein containing the catalytic domain of B23 showed significant but consistently lower activity than VHR in vitro. Among the substrates tested, B23 displayed the highest relative activity toward phosphorylated extracellular signal regulated kinase-1, suggesting that it may be a target for B23 activity in vivo. The B23 transcript was detected in a wide variety of normal human tissues, with relatively high expression in pancreas and brain. B23 was induced by serum stimulation of human fibroblasts as well as by heat shock with similar kinetics to those observed with CL100. Thus, B23 is a new human protein phosphatase which appears to be regulated in response to mitogenic signaling and at least some forms of stress.

Amino Acid Sequence↗
PubMed1994

A human oncogene of the RAS superfamily unmasked by expression cDNA cloning.

As an approach to identify human oncogenes, we generated an expression cDNA library from an ovarian carcinoma line. A potent transforming gene was detected by transfection analysis and identified as TC21, a recently cloned member of the RAS gene superfamily. A single point mutation substituting glutamine for leucine at position 72 was shown to be responsible for activation of transforming properties. While the cDNA clone possessed high transforming activity, the ovarian tumor genomic DNA, which contained the mutated TC21 allele, failed to induce transformed foci. Thus, expression cDNA cloning made it possible to identify and isolate a human oncogene that has evaded detection by conventional approaches.

Amino Acid Sequence↗
PubMed1994

Regulation of keratinocyte growth factor gene expression by interleukin 1.

Keratinocyte growth factor (KGF) is a stromally derived member of the fibroblast growth factor family (FGF7) with potent mitogenic activity on a variety of epithelial cells. To identify molecules that regulate the expression of this paracrine mediator of epithelial proliferation, we investigated the effects of various cytokines and growth factors on KGF production by fibroblasts. The proinflammatory cytokine interleukin 1 (IL1) strongly induced the expression of KGF RNA in fibroblasts from multiple sources. Platelet-derived growth factor BB, IL6, and transforming growth factor alpha caused a moderate elevation, while tumor necrosis factor alpha and basic FGF did not alter the level of KGF RNA expression. The induction by IL1 of KGF transcript levels was time and dose dependent and specifically blocked by anti-IL1 antibodies. Nuclear run on experiments indicated that IL1 stimulated KGF gene transcription. Western blot analysis and keratinocyte proliferation assays demonstrated a corresponding increase in mitogenically active KGF protein in conditioned medium obtained from IL1-treated fibroblasts. The stimulation of KGF expression by IL1 and other cytokines such as IL6, transforming growth factor alpha, and platelet-derived growth factor may provide a mechanism for KGF induction during inflammation that would support its proposed role as mediator of reepithelialization and wound healing.

Adult↗
PubMed1994

Expression complementary DNA library transfer establishes mrp as a multidrug resistance gene.

The emergence of drug-resistant cancer cells is a major obstacle to cancer treatment. Resistant cells often display a multidrug-resistant phenotype that reduces the promise of combination chemotherapy, the classic approach to the prevention of drug resistance. mdr1, a member of the ABC cassette superfamily of transporters which encodes an energy-dependent drug efflux pump, is the only gene known to confer the multidrug-resistant phenotype. Other multidrug resistance mechanisms must exist, since cell lines which have this phenotype in the absence of mdr1 overexpression have been described. We report here the application of a novel approach involving expression complementary DNA library transfer to the identification of drug-resistant genes. Using this approach we establish that mrp, a member of the ABC cassette superfamily of transporters, is capable of conferring a multidrug-resistant phenotype. This approach should be useful in the identification of other novel resistance genes.

3T3 Cells↗
PubMed1994

Keratinocyte growth factor functions in epithelial induction during seminal vesicle development.

Development of the seminal vesicle (SV) is elicited by androgens and is dependent on epithelial-mesenchymal interactions. Androgenic signal transmission from the androgen-receptor-positive mesenchyme to the epithelium has been postulated to involve paracrine factors. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, is produced by stromal/mesenchymal cells and acts specifically on epithelial cells. The KGF transcript was detected by reverse transcription-polymerase chain reaction in newborn mouse SVs and by Northern blot analysis of RNA from cultured neonatal SV mesenchymal cells. Newborn SVs placed in organ culture undergo androgen-dependent growth and differentiation. Addition of a KGF-neutralizing monoclonal antibody to this system caused striking inhibition of both SV growth and branching morphogenesis. This inhibition was due to a decline in epithelial proliferation and differentiation, as the mesenchymal layer was not affected by anti-KGF treatment. When KGF (100 ng/ml) was substituted for testosterone in the culture medium, SV growth was approximately 50% that observed with an optimal dose of testosterone (10(-7) M). All of these findings suggest that KGF is present during a time of active SV morphogenesis and functions as an important mediator of androgen-dependent development.

Animals↗
PubMed1994

High-affinity binding sites for related fibroblast growth factor ligands reside within different receptor immunoglobulin-like domains.

Growth factors of the fibroblast growth factor (FGF) family bind receptors whose external domains are organized in a series of immunoglobulin-like loops. We engineered expression constructs in which cDNAs encoding individual immunoglobulin-like domains of the keratinocyte growth factor (KGF/FGF-7) receptor were fused to the mouse immunoglobulin heavy chain Fc domain (HFc). Each chimera was efficiently secreted from NIH 3T3 transfectants and migrated at the predicted molecular mass after SDS/PAGE. Scatchard analysis revealed that the chimera containing immunoglobulin-like domains 2 (D2) and 3 (D3) bound KGF and acidic FGF at high affinities comparable to the native receptor. However, individual immunoglobulin-like domain chimeras demonstrated marked specificity in their ligand interactions. D2-HFc bound acidic FGF at high affinity, whereas it did not detectably interact with KGF. Conversely, D3-HFc bound KGF at high affinity but exhibited no detectable interaction with acidic FGF. Their selective ligand binding properties were confirmed by the specific neutralization of acidic FGF or KGF mitogenic activity using D2 or D3 HFc, respectively. All of these findings establish that the major binding sites for related FGF ligands are localized to distinct receptor immunoglobulin-like domains.

3T3 Cells↗
PubMed1994

Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product.

The dbl oncogene product contains a 238-amino acid domain, which is shared by an expanding family of growth regulatory proteins. These include the Saccharomyces cerevisiae cell division cycle protein, CDC24, the breakpoint cluster region protein, the ect2 and vav oncogene products, and the brain GDP-releasing factor for Ras. Previous studies have provided evidence that oncogenic Dbl or an associated protein stimulates GDP dissociation from the human species (Hs) homolog of CDC42. We show here that Dbl specifically complexes with the GDP-bound forms of CDC42Hs and RhoA, but not Rac1 or TC10, and that this specificity correlates with the ability of Dbl to act as a GDP-releasing factor. Small deletions throughout the Dbl domain, which inactivate transformation, eliminated the ability of Dbl to stimulate GDP dissociation, whereas deletions outside of this domain did not impair either function. Finally, the Dbl domain itself, when expressed and purified as a recombinant protein, was shown to stimulate GDP dissociation from purified, recombinant CDC42Hs. These findings establish that a minimal unit on Dbl that is critical to its transforming function directly regulates GDP-GTP exchange activity.

3T3 Cells↗
PubMed1994

Keratinocyte growth factor and fibroblast growth factor action on DNA synthesis in rat and human hepatocytes: modulation by heparin.

Keratinocyte growth factor (KGF/FGF-7) is a member of the fibroblast growth factor (FGF) superfamily. Unlike other members of the family, the biological activity of KGF appears to be restricted to epithelial cells. Here we have tested the activity of KGF, acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) on normal adult rat and human hepatocytes and their modulation by heparin. Although more modest than the growth response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF), recombinant KGF enhanced DNA synthesis in rat hepatocytes by two- to threefold. This stimulation occurred in the absence of serum and of other exogenous growth factors. Addition of heparin inhibited the KGF response. Although basic FGF showed little activity on rat hepatocytes, acidic FGF stimulated DNA synthesis by approximately twofold and was substantially enhanced by heparin. In contrast to rat cells, human hepatocytes consistently failed to respond to KGF, aFGF, or bFGF with or without heparin, under conditions where EGF and HGF stimulated DNA synthesis up to sixfold. These results indicate that KGF is capable of acting as a complete mitogen for rat hepatocytes in culture and that the activity is consistent with expression by these cells of a type II FGF receptor subtype, the KGF receptor. These observations suggest that KGF/aFGF together with proteoglycans may help regulate rat but not human liver growth.

Adult↗
PubMed1994

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.

Animals↗
PubMed1994

Prokaryotic expression cloning of a novel human tyrosine kinase.

Screening of a human embryonic lung fibroblast cDNA expression library with antiphosphotyrosine antibodies led to isolation of a novel protein kinase. A clone, designated A6, contained a 3-kb cDNA insert with a predicted open reading frame of 350 amino acids. DNA sequence analysis failed to reveal any detectable similarity with previously known genes, and the predicted A6 protein lacked any of the motifs commonly conserved in the catalytic domains of protein kinases. However, the bacterially expressed beta-galactosidase-A6 fusion protein demonstrated both tyrosine and serine phosphorylation in an in vitro kinase assay and phosphorylated exogenous substrates including myelin basic protein specifically on tyrosine residues. The enzyme also displayed biochemical properties analogous to those of other protein tyrosine kinases. The A6 gene was found to be expressed widely at the transcript level in normal tissues and was evolutionarily conserved. Thus, A6 represents a novel tyrosine kinase which is highly divergent from previously described members of this important class of regulatory molecules.

Amino Acid Sequence↗
PubMed1994

Oncogenic activation of human R-ras by point mutations analogous to those of prototype H-ras oncogenes.

R-ras, K-rev-1/rap and TC21, are more closely related to prototype H-ras than any other known members of the ras superfamily. We recently isolated a mutationally activated TC21 oncogene from a human ovarian carcinoma cell line. Based upon these observations, we sought to re-examine the transforming potential of R-ras, which was reported earlier to lack transforming capacity. Mutations were introduced into the R-ras gene at codons 38 or 87, analogous to positions 12 and 61, respectively, responsible for H-ras oncogene activation. While both mutations resulted in acquisition of R-ras transforming capacity for NIH3T3 cells the position 61 was shown to be more active. Transfectants expressing either R-ras mutant formed colonies in soft agar and were tumorigenic in vivo. As has been reported for H-ras, R-ras cooperated with c-raf-1 in inducing transformation of NIH3T3 cells. These results imply interactions in R-ras and c-raf-1 signaling pathways. We observed R-ras transcripts of 4.6 and 1.2 kb ubiquitously expressed in each of a variety of tissues examined. All these findings raise the possibility that R-ras, like prototype ras genes, may be mutationally activated as an oncogene in some human malignancies.

3T3 Cells↗
PubMed1993

Platelet-derived growth factor AB heterodimer interchain interactions influence secretion as well as receptor binding and activation.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimer comprised of two related polypeptide chains. To investigate the effects of an inactivating lesion introduced into one chain of the nascent PDGF dimer, approaches were developed to optimize synthesis, assembly, secretion, and purification of heterodimers between normal PDGF A and wild-type or mutant PDGF B. PDGF AB heterodimers were released into culture fluids less efficiently than PDGF AA, but to a greater degree than the cell-associated PDGF BB. These results suggest that interactions between two chains influence PDGF secretion. Analysis of heterodimers between PDGF A and disabled PDGF B mutants on cells that express either alpha or beta PDGFRs demonstrated that the impaired biologic activity of the mutant PDGF B chain was ameliorated with respect to binding and triggering of alpha PDGFRs. In cells that expressed both receptor types, heterodimers of mutant PDGF B and wild-type PDGF A gained substantially in their ability to recruit and trigger alpha, but not beta, PDGFRs. Partial rescue of impaired PDGF B mutant chain function by dimer formation with a wild-type PDGF A chain implies that interchain interactions markedly affect PDGFR binding and activation.

3T3 Cells↗
PubMed1993

IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.

Although several interleukin-3 (IL-3)-dependent cell lines proliferate in response to IL-4 or insulin, the 32D line does not. Insulin and IL-4 sensitivity was restored to 32D cells by expression of IRS-1, the principal substrate of the insulin receptor. Although 32D cells possessed receptors for both factors, they lacked the IRS-1--related protein, 4PS, which becomes phosphorylated by tyrosine in insulin- or IL-4--responsive lines after stimulation. These results indicate that factors that bind unrelated receptors can use similar mitogenic signaling pathways in hematopoietic cells and that 4PS and IRS-1 are functionally similar proteins that are essential for insulin- and IL-4--induced proliferation.

Animals↗
PubMed1993

Spleen-derived growth factor, SDGF-3, is identified as keratinocyte growth factor (KGF).

A heparin-binding mitogen rat for rat hepatocytes was partially purified from bovine spleen by a combination of heparin-affinity, cation-exchange and gel-filtration chromatography. Besides stimulating rat hepatocytes, this factor, which was designated spleen-derived growth factor-3 (SDGF-3), exhibited mitogenic activity for mouse epidermal keratinocytes but not mouse fibroblasts. Its apparent epithelial specificity and heparin-binding properties corresponded to those of keratinocyte growth factor (KGF). These findings, together with the fact that the mitogenic activity of SDGF-3 was abolished by a neutralizing monoclonal antibody specific for KGF, identify this bovine spleen-derived hepatocyte mitogen as KGF.

3T3 Cells↗