Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.

BACKGROUND: The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response. MATERIALS AND METHODS: The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines. RESULTS: A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA. CONCLUSION: High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.

Novel yeast-based strategy unveils antagonist binding regions on the nuclear xenobiotic receptor PXR.

BACKGROUND: Ketoconazole binds to and antagonizes pregnane X receptor (PXR) activation. RESULTS: Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. CONCLUSION: Ketoconazole genetically interacts with specific PXR surface residues. SIGNIFICANCE: A yeast-based genetic method to discover novel nuclear receptor interactions with ligands that associate with surface binding sites is suggested. The pregnane X receptor (PXR) is a master regulator of xenobiotic metabolism, and its activity is critical toward understanding the pathophysiology of several diseases, including inflammation, cancer, and steatosis. Previous studies have demonstrated that ketoconazole binds to ligand-activated PXR and antagonizes receptor control of gene expression. Structure-function as well as computational docking analysis suggested a putative binding region containing critical charge clamp residues Gln-272, and Phe-264 on the AF-2 surface of PXR. To define the antagonist binding surface(s) of PXR, we developed a novel assay to identify key amino acid residues on PXR based on a yeast two-hybrid screen that examined mutant forms of PXR. This screen identified multiple "gain-of-function" mutants that were "resistant" to the PXR antagonist effects of ketoconazole. We then compared our screen results identifying key PXR residues to those predicted by computational methods. Of 15 potential or putative binding residues based on docking, we identified three residues in the yeast screen that were then systematically verified to functionally interact with ketoconazole using mammalian assays. Among the residues confirmed by our study was Ser-208, which is on the opposite side of the protein from the AF-2 region critical for receptor regulation. The identification of new locations for antagonist binding on the surface or buried in PXR indicates novel aspects to the mechanism of receptor antagonism. These results significantly expand our understanding of antagonist binding sites on the surface of PXR and suggest new avenues to regulate this receptor for clinical applications.

Cellular processes of v-Src transformation revealed by gene profiling of primary cells--implications for human cancer.

BACKGROUND: Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. In this study, we took advantage of several strains of the Rous sarcoma virus (RSV) to characterize the patterns of v-Src-dependent gene expression in two different primary cell types, namely chicken embryo fibroblasts (CEF) and chicken neuroretinal (CNR) cells. We identified a common set of v-Src regulated genes and assessed if their expression is associated with disease-free survival using several independent human tumor data sets. METHODS: CEF and CNR cells were infected with transforming, non-transforming, and temperature sensitive mutants of RSV to identify the patterns of gene expression in response to v-Src-transformation. Microarray analysis was used to measure changes in gene expression and to define a common set of v-Src regulated genes (CSR genes) in CEF and CNR cells. A clustering enrichment regime using the CSR genes and two independent breast tumor data-sets was used to identify a 42-gene aggressive tumor gene signature. The aggressive gene signature was tested for its prognostic value by conducting survival analyses on six additional tumor data sets. RESULTS: The analysis of CEF and CNR cells revealed that cell transformation by v-Src alters the expression of 6% of the protein coding genes of the genome. A common set of 175 v-Src regulated genes (CSR genes) was regulated in both CEF and CNR cells. Within the CSR gene set, a group of 42 v-Src inducible genes was associated with reduced disease- and metastasis-free survival in several independent patient cohorts with breast or lung cancer. Gene classes represented within this group include DNA replication, cell cycle, the DNA damage and stress responses, and blood vessel morphogenesis. CONCLUSION: By studying the v-Src-dependent changes in gene expression in two types of primary cells, we identified a set of 42 inducible genes associated with poor prognosis in breast and lung cancer. The identification of these genes provides a set of biomarkers of aggressive tumor behavior and a framework for the study of cancer cells characterized by elevated Src kinase activity.

SRC family kinases are required for limb trajectory selection by spinal motor axons.

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chick LMC axons using in ovo electroporation resulted in LMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chick LMC neurons which led to the redirection of LMC axons. Finally, Csk-mediated SFK inhibition attenuated the retargeting of LMC axons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.

Src-Induced cisplatin resistance mediated by cell-to-cell communication.

Cisplatin-induced cell death can be triggered by cell-to-cell communication through gap junctions. Here, we show that activated src produces tyrosine phosphorylation of the gap junction protein connexin 43, decreases gap junction communication, and increases cell survival in response to cisplatin. Experiments with mixed cell populations show that src activity in one cell can confer increased cisplatin survival on neighboring cells, even when the neighboring cells lack such src activity. This work is the first demonstration that expression of an oncogene in one cell can affect the survival of a neighboring cell not expressing the oncogene in response to a chemotherapeutic drug. The trans-acting effect of activated src on neighboring cells can be blocked by inhibitors of src kinase or by siRNA-mediated knockdown of src expression, and it can be counteracted by forced up-regulation of connexin 43, via either gene transfer or proteasome inhibition. These results identify a novel pathway of cisplatin resistance that may be amenable to therapeutic intervention.

Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth.

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool-1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v-Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.

Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system.

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST (glutathione S-transferase)-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl beta-D-thiogalactopyranoside (IPTG)-regulated expression, and the fused protein was purified using GSH (glutathione) affinity chromatography. ELISA (enzyme-linked immunosorbent assay) was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.

Malignant transformation but not normal cell growth depends on signal transducer and activator of transcription 3.

Signal transducer and activator of transcription 3 (STAT3) has been indirectly implicated in numerous fundamental cellular processes, including proliferation, survival, and differentiation. We provide genetic evidence from studies of STAT3-null cells that STAT3 is dispensable for normal growth of mouse fibroblasts in culture. STAT3 contributed to the full induction of some (typified by c-fos) but not all (typified by c-myc) immediate early gene expression, but STAT3-independent processes were sufficient to support full cell growth and survival. However, STAT3 was required to manifest a transformed state following expression of v-src, and STAT3-null cells were impaired for anchorage-independent growth as colonies in soft agar and as tumors in mice. The data suggest that STAT3 mediates the maintenance of focal adhesion kinase activity in the absence of cell adhesion by suppressing the action of an inhibitory phosphatase.

Efficient synthesis and photodynamic activity of porphyrin-saccharide conjugates: targeting and incapacitating cancer cells.

Since the role of saccharides in cell recognition, metabolism, and cell labeling is well-established, the conjugation of saccharides to drugs is an active area of research. Thus, one goal in the use of saccharide-drug conjugates is to impart a greater specificity toward a given cell type or other targets. Although widely used to treat some cancers and age related macular degeneration, the drugs used in photodynamic therapy (PDT) display poor chemical selectivity toward the intended targets, and uptake by cells most likely arises from passive, diffusional processes. Instead, the specific irradiation of the target tissues, and the formation of the toxic species in situ, are the primary factors that modulate the selectivity in the present mode of PDT. We report herein a two-step method to make nonhydrolyzable saccharide-porphyrin conjugates in high yields using a tetra(pentafluorophenyl)porphyrin and the thio derivative of the sugar. As a demonstration of their properties, the selective uptake (and/or binding) of these compounds to several cancer cell types was examined, followed by an investigation of their photodynamic properties. As expected, different malignant cell types take up one type of saccharide-porphyrin conjugate preferentially over others; for example, human breast cancer cells (MDA-MB-231) absorb a tetraglucose-porphyrin conjugate over the corresponding galactose derivative. Doseametric studies reveal that these saccharide-porphyrin conjugates exhibit varying PDT responses depending on drug concentration and irradiation energy. (1) Using 20 microM conjugate and greater irradiation energy induces cell death by necrosis. (2) When 10-20 microM conjugate and less irradiation energy are used, both necrosis and apoptosis are observed. (3) Using 10 microM and the least irradiation energy, a significant reduction in cell migration is observed, which indicates a reduction in aggressiveness of the cancer cells.

Expression of protein tyrosine kinase pp60v-src variants in Dictyostelium discoideum.

We achieved production of v-Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo.

In vitro transformation of primary cultures of chick embryo fibroblasts by the membrane-bound vSrc or the nuclear vJun oncoproteins is correlated with a down-regulation of the secreted glycoprotein SPARC (also called BM-40 or osteonectin). This protein is a nonstructural component of the extracellular matrix that is thought to regulate cell-matrix interaction during development, wound repair, and carcinogenesis. Its precise function remains unclear. To estimate the contribution of SPARC down-regulation to the major aspects of the transformed phenotype, we have reexpressed this protein from a self-replicating retrovirus Rcas, designated R-SPARC, in the transformed cultures. These R-SPARC-infected cultures display the following main properties: (i) they accumulate the SPARC protein to a level identical to or only slightly higher than the level in normal chick embryo fibroblasts; (ii) they retain the main phenotypic properties characteristic of in vitro transformed cells, that is, altered morphology, capacity to grow in a reduced amount of serum, and capacity to develop colonies from single cells in agar; (iii) they display a clearly reduced capacity to develop local fibrosarcomas in vivo. Taken together, these data strongly suggest that down-regulation of SPARC contributes to the transformed phenotype triggered by vSrc and vJun in primary avian fibroblasts, by facilitating in vivo tumorigenesis.

Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src.

Two eps8 isoforms, p97eps8 and p68eps8, were previously identified as substrates for receptor tyrosine kinases. Analysis of eps8 phosphotyrosine content in v-Src transformed cells (IV5) revealed that both isoforms were highly tyrosyl phosphorylated and their readiness to be phosphorylated by Src in vitro further indicated that they were putative Src substrates as well. Indeed, the enhancement of tyrosyl phosphorylation of p97eps8 detected in cells coexpressing both p97eps8 and active Src relative to that in cells expressing p97eps8 alone supported our hypothesis. The existence of common phosphotryptic peptides between in vitro 32P-labeled p97eps8 and p68eps8 indicated that these two proteins shared the same Src-mediated sites. Further in vitro binding assays demonstrated that p68eps8 was the major eps8 isoforms that could be precipitated by bacterial fusion protein containing Src SH3. Interestingly, both p68eps8 and p97eps8 were preferentially expressed in v-Src transformed cells and the presence of p68eps8 appeared to depend on Src. Since p97eps8 has been implicated in mitogenesis and tumorigenesis, its readiness to be phosphorylated and induced by v-Src might attribute to v-Src-mediated transformation.

v-src induces cisplatin resistance by increasing the repair of cisplatin-DNA interstrand cross-links in human gallbladder adenocarcinoma cells.

Activation of Src, which has an intrinsic protein tyrosine kinase (PTK) activity, has been demonstrated in human solid tumors, such as colorectal and breast cancers. To investigate the role of activated Src in drug resistance, we evaluated the effect of v-src on the resistance to various anti-cancer drugs using v-src-transfected HAG-1 human gallbladder adenocarcinoma cells. Compared with parental or mock-transfected HAG-1 cells, v-src-transfected HAG/src3-1 cells showed a 3.5-fold resistance to cis-diamminedichloroplatinum (II) (CDDP) but not to doxorubicin, etoposide or 5-fluorouracil. By contrast, activated H-ras, which acts downstream of src, failed to induce resistance to either of these drugs. Furthermore, wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, and H7, a protein kinase C (PKC) inhibitor, did not alter CDDP resistance. Evaluation of the kinetics of the removal of DNA interstrand cross-links (ICLs), measured by alkaline elution, showed a significant increase in this removal in HAG/src3-1 cells as compared with mock-transfected cells, though no differences were found in the formation of DNA ICLs between these cell lines. CDDP resistance in v-src-transfected cells was reversed, if not completely, by either herbimycin A or radicicol, specific inhibitors of Src-family PTKs, suggesting that Src tyrosine kinase activity induces CDDP resistance. Moreover, significant reduction in the repair of CDDP-induced DNA ICLs was observed upon treatment with radicicol. The intracellular glutathione content and mRNA expression of topoisomerase II and metallothionein were virtually identical between these cell lines, except for topoisomerase I mRNA. Our data strongly suggest that the ability of activated src, but not ras, to induce CDDP resistance is mediated by augmentation of DNA repair through Src to downstream signal-transduction pathways distinct from either the Ras, PI 3-kinase or PKC pathway.

1a-docosahexaenoyl mitomycin C: a novel inhibitor of protein tyrosine kinase.

A series of derivatives of mitomycin C conjugated with various fatty acids at position 1a was synthesized and the effect of these compounds on protein kinase activities was evaluated. 1a-Docosahexaenoyl mitomycin C (DMMC) selectively inhibited protein tyrosine kinase (PTK) activity in the postnuclear fraction of v-src-transformed NIH 3T3 cells although neither derivatives conjugated with other fatty acids or docosahexaenoic acid or mitomycin C did not. DMMC inhibited the activity of calmodulin-dependent kinase III and protein kinase A very weakly, and only barely affected protein kinase C activity. DMMC also attenuated autophosphorylation of immunoprecipitated p60v-src irreversibly. The addition of thiol compounds to the reaction mixture reversed the inhibition by DMMC, suggesting that some thiol moiety of PTK protein might be involved. DMMC also inhibited kinase activity of p210bcr-abl immunoprecipitated from the lysate of K562 cells. These results indicate that DMMC is a novel inhibitor of PTK.

Cell adhesion and the integrin-linked kinase regulate the LEF-1 and beta-catenin signaling pathways.

The integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase that can interact directly with the cytoplasmic domains of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. Overexpression of constitutively active ILK results in loss of cell-cell adhesion, anchorage-independent growth, and tumorigenicity in nude mice. We now show that modest overexpression of ILK in intestinal epithelial cells as well as in mammary epithelial cells results in an invasive phenotype concomitant with a down-regulation of E-cadherin expression, translocation of beta-catenin to the nucleus, formation of a complex between beta-catenin and the high mobility group transcription factor, LEF-1, and transcriptional activation by this LEF-1/beta-catenin complex. We also find that LEF-1 protein expression is rapidly modulated by cell detachment from the extracellular matrix, and that LEF-1 protein levels are constitutively up-regulated at ILK overexpression. These effects are specific for ILK, because transformation by activated H-ras or v-src oncogenes do not result in the activation of LEF-1/beta-catenin. The results demonstrate that the oncogenic properties of ILK involve activation of the LEF-1/beta-catenin signaling pathway, and also suggest ILK-mediated cross-talk between cell-matrix interactions and cell-cell adhesion as well as components of the Wnt signaling pathway.

V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.

Adaptation to hypoxia represents an important aspect of tumor progression. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor (VEGF). In this report, we demonstrate that whereas C-SRC expression is not required for expression of HIF-1 or transcriptional activation of genes encoding VEGF and enolase 1 (ENO1), cells expressing the v-Src oncogene have increased expression of HIF-1, VEGF, and ENO1 under both hypoxic and nonhypoxic conditions. Expression of V-SRC was associated with increased transcription of reporter genes containing cis-acting hypoxia-response elements from the VEGF and ENO1 genes, and this transcriptional activation required an intact HIF-1 binding site. When three rat hepatoma subclones that differed with respect to the level of HIF-1 expression were injected into nude mice, tumor growth correlated with HIF-1 expression, suggesting that HIF-1 may be generally involved in tumor progression. These studies link an oncogene to the induction of HIF-1 expression, thus providing a mechanism for hypoxic adaptation by tumor cells.

The role of the Src homology domains in morphological transformation by v-src.

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.

Altered expression of src tyrosine kinase after axotomy.

Although most peripheral neurons show regenerative changes after axotomy, neurons of some nuclei show degenerative changes. To study the mechanisms responsible for such differences in neuronal reactions after axotomy, we investigated the changes of pp60c-src and phosphotyrosine (pTyr) in the dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus after transection of each nerve in adult rats using immunohistochemistry. Polyclonal antibody against pp60v-src stained the cytoplasm of neurons homogeneously in both the severed and non-severed sides of the vagal and hypoglossal nuclei from days 1 to 56 after axotomy. On the other hand, the monoclonal antibody against pp60v-src (mAb327, which recognizes src homology 3 region in pp60src as an epitope) showed different immunoreactivities. No pp60c-src immunoreactivity was observed in neurons in either the vagal and hypoglossal nuclei of the non-severed side between days 1 and 56, whereas in the severed side of the hypoglossal nucleus intense pp60c-src immunoreactivity was observed along the plasma membrane of neurons from days 5 to 28 after axotomy. In contrast, in the severed side of the vagal nucleus pp60c-src immunoreactivity appeared along the plasma membrane of neurons on day 5 and remained until day 7. Conformational changes accompanying the activation of pp60c-src are suggested to cause differences in immunoreactivities of these antibodies. The expression (or activation) of pp60c-src in the neurons was stronger and longer lasting in the hypoglossal nucleus, which regenerates successfully after axotomy, than in the dorsal motor nucleus of the vagus nerve, which undergoes necrobiotic reaction. These results indicate that pp60c-src plays an important role in regeneration after axotomy.

Constitutive activation of JAK1 in Src-transformed cells.

We have previously found that the signal transducer and activator of transcription (Stat) 3 is constitutively activated in cells stably transformed by the v-Src oncoprotein. While activation of Stat proteins has also been observed following epidermal growth factor or platelet-derived growth factor stimulation, Stat3 activation is more commonly associated with signaling through cytokine receptors and activation of the Janus family tyrosine kinases JAK1 or JAK2. We therefore investigated whether JAK1 or JAK2 were activated in Src-transformed cells. In three v-Src-transformed fibroblast cell lines (NIH3T3, Balb/c, and 3Y1), JAK1 displayed increased tyrosyl phosphorylation compared to non-transformed cells. The level of tyrosyl phosphorylation of JAK1 was significantly greater in NIH3T3 cells transformed by expression of v-Src or high levels of a constitutively active mutant of c-Src (Y527F) than in cells overexpressing the less transforming normal c-Src. Enzymatic activity of JAK1 was assessed using autophosphorylation assays. In anti-JAK1 immunoprecipitates from v-Src-transformed NIH3T3 cells, a protein with the same migration as JAK1 showed substantially increased levels of 32P incorporation compared to immunoprecipitates from non-transformed cells. Similar results were obtained using anti-JAK2 immunoprecipitates; however, the level of JAK2 tyrosyl phosphorylation and 32P incorporation in anti-JAK2 immunoprecipitates were markedly lower than in anti-JAK1 immunoprecipitates. We conclude that JAK1, and possibly JAK2, are constitutively activated in Src-transformed cells, raising the possibility that Janus family kinases contribute to the constitutive activation of Stat3 previously observed in these cells and/or other properties of Src-transformed cells.

v-src interferes with the in vitro erythropoietic stimulatory ability of spleen stromal cells through repression of vascular cell adhesion molecule-1 and stem cell factor.

A mouse spleen stromal cell line, MSS62, can create an in vitro erythropoietic microenvironment in which development of erythropoietin-responsive progentor cells is stimulated by cell-cell contact via stem cell factor (SCF)/c-Kit and vascular cell adhesion molecule-1 (VCAM-1)/very late activation antigen-4 (VLA-4) interactions between stromal and erythroid cells. To find out the effect of src on the erythropoietic microenvironment, MSS62 cells were transfected with v-src oncogene, and its effect on erythropoietic stimulatory activity was measured. Transfectants with high v-Src activity showed reduction in erythropoietic stimulatory activity. A decrease in cell-surface VCAM-1 and SCF mRNA was accompanied by high v-Src activity. These results suggest that v-Src interferes with the erythropoietic stimulatory activity of the stromal cells through repression of VCAM-1 and SCF.