Overexpression of Csk-binding protein contributes to renal cell carcinogenesis.

C-terminal Src kinase (Csk)-binding protein (Cbp) is a transmembrane adaptor protein that localizes exclusively in lipid rafts, where it regulates Src family kinase (SFK) activities through recruitment of Csk. Although SFKs are well known for their involvement in cancer, the function of Cbp in carcinogenesis remains largely unknown. In this study, we reported overexpression of Cbp in more than 70% of renal cell carcinoma (RCC) specimens and in the majority of tested RCC cell lines. Depletion of Cbp in RCC cells by RNA interference led to remarkable inhibition of cell proliferation, migration, anchorage-independent growth as well as tumorigenicity in nude mice. Strikingly, silencing of Cbp negatively affected the sustaining of Erk1/2 activation but not c-Src activation induced by serum. Besides, the RhoA activity in RCC cells was remarkably impaired when Cbp was knocked down. Overexpression of wild-type Cbp, but not its mutant Cbp/DeltaCP lacking C-terminal PDZ-binding motif, significantly enhanced RhoA activation and cell migration of RCC cells. These results provided new insights into the function of Cbp in modulating RhoA activation, by which Cbp might contribute to renal cell carcinogenesis.

Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.

Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation.

Epidermal growth factor receptor (EGFR) and Src tyrosine kinase cooperate in regulating EGFR-mediated cell signaling and promoting cell transformation and tumorigenesis in pathological conditions. Activation of Src is tightly regulated by the C-terminal Src kinase (Csk). The Csk-binding protein (Cbp) is a ubiquitously expressed transmembrane protein. Its functions include suppression of T-cell receptor activation through recruiting Csk and inhibiting Src family kinase (SFK). However, a potential role of Cbp in EGF-induced cell activities has not been investigated. Here, we report that EGF-stimulation-induced Cbp tyrosine phosphorylation followed by Cbp-Csk association, in a SFK-dependent manner. Expression of wild-type (wt) Cbp remarkably suppressed EGF-induced activation of Src, ERK1/2, and Akt-1 enzymes, and NIH3T3 cell transformation, as well as colony formation of a breast cancer cell line (MDA-MB-468) in soft agar. In contrast, expression of CbpY317F or knockdown endogenous Cbp in NIH3T3 cells by RNA interference significantly enhanced EGF-induced activation of these enzymes and cell transformation. In addition, overexpression of multiple receptor tyrosine kinases (RTKs)-induced Cbp tyrosine phosphorylation. These results demonstrate that Cbp functions as a negative regulator of cell transformation and tumor cell growth through downregulation of Src activation, suggesting that Cbp might be broadly involved in RTKs-activated signaling pathways and tumorigenesis.

A novel role for protein tyrosine phosphatase shp1 in controlling glial activation in the normal and injured nervous system.

Tyrosine phosphorylation regulated by protein tyrosine kinases and phosphatases plays an important role in the activation of glial cells. Here we examined the expression of intracellular protein tyrosine phosphatase SHP1 in the normal and injured adult rat and mouse CNS. Our study showed that in the intact CNS, SHP1 was expressed in astrocytes as well as in pyramidal cells in hippocampus and cortex. Axotomy of peripheral nerves and direct cortical lesion led to a massive upregulation of SHP1 in activated microglia and astrocytes, whereas the neuronal expression of SHP1 was not affected. In vitro experiments revealed that in astrocytes, SHP1 associates with epidermal growth factor (EGF)-receptor, whereas in microglia, SHP1 associates with colony-stimulating factor (CSF)-1-receptor. In postnatal and adult moth-eaten viable (me(v)/me(v)) mice, which are characterized by reduced SHP1 activity, a strong increase in reactive astrocytes, defined by GFAP immunoreactivity, was observed throughout the intact CNS, whereas neither the morphology nor the number of microglial cells appeared modified. Absence of (3)[H]-thymidine-labeled nuclei indicated that astrocytic proliferation does not occur. In response to injury, cell number as well as proliferation of microglia were reduced in me(v)/me(v) mice, whereas the posttraumatic astrocytic reaction did not differ from wild-type littermates. The majority of activated microglia in mutant mice showed rounded and ameboid morphology. However, the regeneration rate after facial nerve injury in me(v)/me(v) mice was similar to that in wild-type littermates. These results emphasize that SHP1 as a part of different signaling pathways plays an important role in the global regulation of astrocytic and microglial activation in the normal and injured CNS.

Characteristics of EGFR family-mediated HRG signals in human ovarian cancer.

The ability of the epidermal growth factor receptor (EGFR) family members, EGFR, HER2, HER3, and HER4, to form homo- and heterodimers after interaction with different ligands expands the signal diversity of these proteins. We investigated their mechanism of activation by exogenous EGF and heregulin (HRG) in human ovarian carcinoma cell lines which express different amounts and combinations of the four receptors. Consistently the predominant interaction after EGF treatment was between EGFR and HER2, whereas activation of HER3 and HER4 depended on the relative abundance of the four receptors in the cells. Remarkably HER3 activation by HRG could occurs independent of HER2, and in one cell line almost no HER4 activation by HRG was detected despite high levels expression. Both EGF and HRG induced activation of mitogen-activated protein kinase (MAPK), but the time course of MAPK activation differed depending on the hetero-dimers induced. EGF and HRG mediated cell growth through the EGFR/HER2 heterodimer and HER4, respectively, but not through HER3 when it was the only HRG receptor expressed and phosphorylated in the cells. These findings reveal a distinct pattern of HRG induced EGFR family interaction in ovarian cancer that is distinct from that described in human breast cancer. Moreover EGF and HRG can exert distinct biological functions depending on the receptor complexes induced in a given ovarian cancer cell line.

The receptor-like protein-tyrosine phosphatase DEP-1 is constitutively associated with a 64-kDa protein serine/threonine kinase.

Protein-tyrosine phosphatases (PTPs) are involved in the regulation of diverse cellular processes and may function as positive effectors as well as negative regulators of intracellular signaling. Recent data demonstrate that malignant transformation of cells is frequently associated with changes in PTP expression or activity. Our analysis of PTP expression in mammary carcinoma cell lines resulted in the molecular cloning of a receptor-like PTP, also known as DEP-1. DEP-1 was found to be expressed at varying levels in mammary carcinoma cell lines and A431 cells. In all tumor cell lines analyzed, DEP-1 was constitutively phosphorylated on tyrosine residues. Phosphorylation of DEP-1 increased significantly after treatment of cells with the PTP inhibitor pervanadate. In A431 cells, tyrosine phosphorylation of DEP-1 was also observed after stimulation with epidermal growth factor, however, only after prolonged exposure of the cells to the ligand, suggesting an indirect mechanism of phosphorylation. In addition, DEP-1 coprecipitated with several tyrosine-phosphorylated proteins from pervanadate-treated cells. In vitro binding experiments using a glutathione S-transferase fusion protein containing the catalytically inactive PTP domain of DEP-1 (Gst-DEP-1-C/S) identify these proteins as potential substrates of DEP-1. In addition, we found a 64-kDa serine/threonine kinase to be constitutively associated with DEP-1 in all tumor cell lines tested. The 64-kDa kinase forms a stable complex with DEP-1 and phosphorylates DEP-1 and DEP-1-interacting proteins in vitro. These data suggest a possible mechanism of DEP-1 regulation in tumor cell lines involving serine/threonine and/or tyrosine phosphorylation.

Adapter function of protein-tyrosine phosphatase 1D in insulin receptor/insulin receptor substrate-1 interaction.

Insulin signal transduction involves the multisite docking protein insulin receptor substrate-1 (IRS-1) and a number of Src homology-2 (SH2) domain factors, including p85/p110 phosphatidylinositol 3-kinase, p110 GTPase-activating protein, and the phosphotyrosine-specific phosphatase PTP1D. In transfected baby hamster kidney cells, Rat1 fibroblasts, and normal IM9 lymphoblasts, PTP1D directly binds activated insulin receptor. This interaction is mediated by catalytic domain-proximal SH2 determinants of the phosphatase and phosphotyrosine 1146 of the activated insulin receptor. While the receptor and the phosphatase do not serve as substrates for each other, their interaction promotes IRS-1 binding to the receptor, indicating that PTP1D functions as an adapter for insulin receptor and IRS-1. The formation of a multiprotein signaling complex involving the insulin receptor, PTP1D, and IRS-1 enhances cellular glucose uptake, a critical process in the physiological action of insulin.

Amplification of HER-2(erbB-2/neu) oncogene as the most significant prognostic factor in a group of Russian breast cancer patients.

Amplification of HER-2(erbB-2/neu) oncogene was detected in 36 of 142 (25%) breast carcinomas (BC) RNA expression was examined in 42 carcinomas, in 10 of them overexpression was revealed. Amplification was matched by overexpression. No association was found between the increased number of HER-2(erbB-2/neu) copies and tumor size, lymph node involvement, stage of disease, age of onset, and estrogen and progesterone receptor level. HER-2(erbB-2/neu) amplification was shown to be of independent prognostic significance in the group of 32 BC patients with sufficient follow-up (more than 40 months). Six of 7 HER-2(erbB-2/neu) amplification-positive patients and only 2 of 25 HER-2(erbB-2/neu) amplification-negative ones relapsed (p < 0.00005).

Loss of heterozygosity at chromosome 17p is associated with HER-2 amplification and lack of nodal involvement in breast cancer.

Sixty DNA samples from breast carcinoma (BC) patients were analyzed by Southern blot to examine certain oncogene and anti-oncogene alterations. Amplification of the HER-2 oncogene was detected in 15 tumours (25%), c-myc in 2 (3%) only and HER-1 in none. Distribution of Hras-1 oncogene alleles in BC did not significantly differ from that seen in healthy donors. Not a single case of RB-1 anti-oncogene alteration and only one of p53 suppressor gene abnormality was found when appropriate cDNA copies were used as probes. With the use of DNA polymorphic markers, loss of heterozygosity (LOH) was revealed at chromosome 17p (probe YNZ-22) in 18 of 39 (46%) informative cases, at 17q (probe THH-59) in 10 of 34 (29%) and at 11p (probe Hras-1) in 8 of 30 (27%). The only significant correlation between these genetic alterations and the clinical characteristics of the tumours studied was the association of LOH at 17p with node-negative BC (p < 0.02). Also, the tendency for correlation (p < 0.2) between HER-2 amplification and loss of 17p sequences was revealed: 7 of 10 amplification-positive, but only 11 of 29 amplification-negative BC possessed LOH of YNZ-22 locus.

Amplification of ERBB-2 (HER-2/NEU) oncogene in different neoplasms of patients from USSR.

203 tumor specimens from 175 patients were studied. Amplification of ERBB-2 was detected in 14 out of 63 (22%) cases of breast carcinoma, in 1 out of 23 patients with ovarian cancers, in 1 out of 19 cases of colon carcinoma and in 1 out of 27 patients with thyroid cancer. We failed to find more than one copy of ERBB-2 in 34 patients with lung cancers, 6 with sarcomas and 3 with melanomas. There was tendency toward correlation between ERBB-2 amplification and lymph node involvement in patients with breast carcinoma. Thus, the oncogene ERBB-2 is often amplified in human tumors, but breast cancer is characterized by an especially high frequency of ERBB-2 amplification.

Distribution and rearrangements of alleles of c-Ha-ras-1 protooncogene and their correlation with the development of lung, ovarian and thyroid cancers.

The protooncogene c-Ha-ras-1 locus in 84 cancer patients was examined for allelic restriction fragment length polymorphism. The distribution of four common c-Ha-ras-1 alleles (a1, a2, a3 and a4) in lung, ovarian and thyroid cancer patients was analyzed. In approximately half (8 out of 15) of lung and ovarian carcinomas possessing the a4 allele, alterations of the c-Ha-ras-1 locus (deletion of allele with the shorter fragment length, amplification of a4 allele, change of allele fragment length) were detected as compared to 2 cases of rearrangement out of 40 tumors lacking the a4 allele. An increased a4 allele frequency was found in individuals with lung and ovarian tumors as compared to controls presented in literary data and thyroid cancer patients. On the other hand, homozygosity for the a2 locus resulting from the deletion of another allele, and increased a2 allele frequency in thyroid cancer patients were observed. Thus the a4 and a2 alleles of c-Ha-ras-1 may perhaps be viewed as genetic markers of predisposition to lung, ovarian and thyroid cancer, respectively, in combination with other clinical parameters.

Virus-specific sequences in nuclear DNA from non-producer hamster Rous sarcoma.

Proviral DNA from non-producer Rous sarcoma in Syrian hamster contains practically all the nucleotide sequences presented in 125I-labeled RNA from Rous sarcoma virus, Carr-Zilber strain. Virus-specific sequences consist of moderately reiterated and unique DNA regions. The amount of Rous sarcoma virus-specific provirus equivalents in hamster Rous sarcoma DNA is equal to 5.2 +/- 0.01. Experiments on transfection show that proviral DNA studied possesses biological activity in respect to cell transformation and virus production. The infectivity of DNA from hamster tumor does not depend on the expression of group-specific (gs) antigen in the recipient cells.

Culture of human cells obtained with DNA from chick Rous sarcoma.

An infectious process was reproduced in the culture of chick embryo cells by means of DNA isolated from Rous chick sarcoma tissue (Carr-Zilber strain). This DNA preparation displays biological activity also in the culture of human embryo diploid cells (HEDC) which is manifested in: 1. discontinuous synthesis of avian oncovirus group-specific antigen; 2. enhancement of proliferative activity and morphological transformation of human cells; 3. continuous presence of virus-specific sequences as revealed by DNA/RNA hybridization. Producing complete oncornavirus by means of DNA isolated from Rous chick sarcoma in HEDC was unsuccessful. DNA preparation from gs negative chick embryo cells shows no infectious activity in HEDC culture.