Human Tumor-derived Cell Research Articles

Innately Malignant?

Chien et al . investigated pathways downstream of Ras-related guanosine triphosphatase B (RalB), which has been implicated in Ras-induced oncogenic transformation, and unearthed a link between oncogenesis and the innate immune response. An RNAi screen revealed that, like RalB depletion, depletion of Sec5 (a component of the exocyst complex, which is involved in tethering secretory vesicles to the plasma membrane) induced apoptosis in human tumor-derived cell lines but not in nontumorigenic epithelial cells. Analysis of the exocyst complex revealed the atypical IκB kinase TBK1 (Tank binding kinase 1, a known component of the innate immune response), which bound to Sec5 in in vitro assays and coimmunoprecipitated with it from HEK293 cells; moreover, TBK1 depletion stimulated apoptosis in tumor-derived cells. Mouse embryo fibroblasts (MEFs) lacking TBK1 were more likely than wild-type MEFs to undergo apoptosis in response to oncogenic K-ras and were less likely to undergo oncogenic transformation, whereas expression of catalytically inactive TBK1 in conjunction with oncogenic K-ras promoted apoptosis. A constitutively active form of Ral promoted TBK1 association with Sec5, enhanced TBK1-dependent phosphorylation of IRF-3 (interferon regulatory factor-3), and stimulated Sec5- and TBK1-dependent nuclear translocation of IRF-3. Both polyinosine-polycytidylic acid [poly(I:C), a mimic of viral dsRNA] and Sendai virus activated endogenous RalB in human bronchial epithelial cells; Sec5 depletion inhibited poly(I:C)- and Sendai virus-dependent translocation of IRF-3 and activation of target genes. Drosophila with a single copy of dSec5 or a hypomorphic mutation of dRal showed an attenuated response to fungus. Thus, the RalB-Sec5-TBK1 connection appears to link innate immune signaling to cancer cell survival. Mantovani and Balkwill discuss the results in the context of the known relationship between inflammation and cancer. Y. Chien, S. Kim, R. Bumeister, Y.-M. Loo, S. W. Kwon, C. L. Johnson, M. G. Balakireva, Y. Romeo, L. Kopelovich, M. Gale Jr., C. Yeaman, J. H. Camonis, Y. Zhao, M. A. White, RalB GTPase-mediated activation of the IκB family kinase TBK1 couples innate immune signaling to tumor cell survival. Cell 127 , 157-170 (2006). [PubMed] A. Mantovani, F. Balkwill, RalB signaling: A bridge between inflammation and cancer. Cell 127 , 42-44 (2006). [PubMed]

Dppa2 and Dppa4 Are Closely Linked SAP Motif Genes Restricted to Pluripotent Cells and the Germ Line

Despite the enormous medical potential of ESCs, the molecular mechanisms conferring the ability to differentiate into all cell types of the embryo remain elusive. We used an in silico approach to identify genes expressed exclusively in mouse preimplantation embryos and pluripotent cell lines. Two of these genes were developmental pluripotency-associated gene 2 (Dppa2) and Dppa4, which we show are closely linked genes encoding putative nuclear SAP domain proteins expressed in human and mouse pluripotent stem cells and germ cell tumor-derived embryonal carcinoma cells. In the mouse, these genes are transcribed in germinal vesicle-stage oocytes and throughout the cleavage stages of embryogenesis. They then become restricted to the pluripotent inner cell mass of blastocysts and are subsequently downregulated. After gastrulation, Dppa2 and Dppa4 are expressed only in the developing germ line, showing that these genes mark cells of the pluripotent cycle. In the germ line, both genes are downregulated as the germ cells commit to the oogenic pathway or soon after commitment to the spermatogenic pathway. We have observed similar germ line expression profiles for other pluripotent markers, and these results are consistent with the hypothesis that pluripotent markers must be downregulated during fetal germ line development, a process that may be required to facilitate appropriate germ line differentiation. The study of expression and function of pluripotent markers such as Dppa2 and Dppa4 is likely to unveil new aspects of the regulation of pluripotency and germ line development in mammals.

The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

Tumor-derived endothelial cells (TEC) display increased survival and angiogenic properties in respect to normal endothelial cells. The aim of this study was to investigate the mechanism potentially involved in TEC proangiogenic phenotype. We found that thrombospondin-1 (TSP-1), a potent physiological inhibitor of angiogenesis, was significantly reduced in TEC in respect to normal endothelial cells. This reduction was confirmed by immunofluorescence in the intratumor vessels of clear cell renal carcinomas. As TEC were shown to display a basal upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we evaluated the possible regulation of TSP-1 by this pathway by using LY294002 and wortmannin, the PI3K inhibitors, and rapamycin, the mammalian target of rapamycin (mTOR) inhibitor. In addition, we developed negative dominant TEC for Akt. TSP-1 production by TEC was enhanced by the treatment with LY294002 and wortmannin and with rapamycin, suggesting a negative regulation of TSP-1 expression by the PI3K/Akt/mTOR pathway. In addition, downregulation of Akt activation in negative dominant Akt TEC enhanced TSP-1 expression and release. Administration of exogenous TSP-1 to TEC reduced their proangiogenic properties in vitro and in vivo. In parallel, blockade of TSP-1 with an anti-TSP-1 antibody in negative dominant Akt TEC restored their proangiogenic phenotype to levels similar to wild-type TEC. In conclusion, these results indicate that the upregulation of the PI3K/Akt/mTOR pathway is responsible for the inhibition of TSP-1 synthesis which is critical in determining the proangiogenic phenotype of TEC. Strategies aimed to inhibit the PI3K/Akt/mTOR pathway may restore a normal quiescent endothelial phenotype in TEC by promoting TSP-1 production.

The effect of imatinib mesylate (Glivec) on human tumor-derived cells

Imatinib mesylate is a specific inhibitor of the Bcr-Abl protein tyrosine kinase that competes with ATP for its specific binding site in the kinase domain. It has activity against platelet-derived growth factor receptor alpha and beta (PDGFR-alpha and -beta), and c-kit, the receptor for stem cell factor. We have used a standardized ATP-tumor chemosensitivity assay and immunohistochemistry to determine the cytotoxicity of imatinib mesylate in tumor-derived cells from cutaneous and uveal melanoma, and ovarian carcinoma. Imatinib mesylate was tested at concentrations ranging from 2.0 to 0.0625 micromol/l alone and in combination with a cytotoxic drug (cisplatin, doxorubicin, paclitaxel or treosulfan). Imatinib mesylate showed low inhibition (IndexSUM>300) across the range of concentrations tested in this study, with few tumors exhibiting increasing inhibition with increased drug concentration. The median IC90 values for cutaneous and uveal melanoma and ovarian carcinoma were 13.2 micromol/l (4.0-294.3 micromol/l), 12.0 micromol/l (2.0-285.4 micromol/l) and 7.71 micromol/l (6.51-11.02 micromol/l), respectively. Imatinib mesylate potentiated the effect of different cytotoxics in 9% (5/54) of cases and had a negative effect in 13% (7/54) of cases, with no effect in the remainder. No correlation of effect was noted with c-kit, platelet-derived growth factor receptor-alpha or platelet-derived growth factor receptor-beta expression, assessed by immunohistochemistry. The signaling pathways mediated by activation of c-kit or platelet-derived growth factor receptor may act as antiapoptotic survival signals in some cancers and inhibition of these pathways may potentiate the activity of some cytotoxic drugs by inhibiting the survival signal. Growth inhibition, however, may reduce the efficacy of cytotoxic drugs, which tend to target proliferating cells preferentially, and clinical effects are therefore difficult to predict.

Oxytocin Induces Proliferation and Migration in Immortalized Human Dermal Microvascular Endothelial Cells and Human Breast Tumor-Derived Endothelial Cells

Oxytocin either increases or inhibits cell growth in different cell subtypes. We tested here the effect of oxytocin on cell proliferation and migration of human dermal microvascular endothelial cells (HMEC) and tumor-associated endothelial cells purified from human breast carcinomas (B-TEC). Oxytocin receptors were expressed in both cell subtypes at mRNA and protein levels. Through oxytocin receptor, oxytocin (1 nmol/L-1 mumol/L) significantly increased cell proliferation and migration in both HMEC and B-TEC, and addition of a selective oxytocin antagonist fully reverted these effects. To verify whether a different expression of adhesion molecule-related genes could be responsible for the oxytocin-induced cell migration, untreated and treated cells were compared applying a microarray technique. In HMEC, oxytocin induced the overexpression of the matrix metalloproteinase (MMP)-17, cathepsin D, and integrin beta(6) genes. In B-TEC, oxytocin significantly switched on the gene profile of some MMP (MMP-11 and MMP-26) and of integrin beta(6). The up-regulation of the integrin beta(6) gene could be involved in the oxytocin-induced cell growth, because this subunit is known to determine activation of mitogen-activated protein kinase-extracellular signal-regulated kinase 2, which is involved in the oxytocin mitogenic effect. In B-TEC, oxytocin also increased the expression of caveolin-1 at gene and protein levels. Because oxytocin receptor localization within caveolin-1-enriched membrane domains is necessary for activation of the proliferative (instead of the inhibitory) response to oxytocin, its enhanced expression can be involved in the oxytocin-induced B-TEC growth as well. Altogether, these data indicate that oxytocin contributes to cell motility and growth in HMEC and B-TEC.

Expression of Pax2 in Human Renal Tumor-Derived Endothelial Cells Sustains Apoptosis Resistance and Angiogenesis

The transcription factor Pax2 is known to play a key role during renal development and to act as an oncogene favoring renal tumor growth. We recently showed that endothelial cells derived from human renal carcinomas display abnormal characteristics of survival and angiogenic properties. In the present study we found that renal tumor-derived endothelial cells, but not normal endothelial cells, expressed Pax2 protein and mRNA. To down-regulate Pax2 expression, we transfected tumor-derived endothelial cells with an anti-sense PAX2 vector whereas we transfected normal human microvascular endothelial cells with a sense PAX2 vector to induce Pax2 expression. The inhibition of Pax2 expression in tumor-derived endothelial cells induced an increase in tumor suppressor PTEN expression and a decrease in Akt phosphorylation. In addition, decreased apoptosis resistance, adhesion, invasion, and in vitro and in vivo angiogenesis were observed. Conversely, Pax2 induction in normal endothelial cells conferred to these cells a proinvasive, proangiogenic phenotype similar to that of tumor-derived endothelial cells. These results indicate that Pax2 is involved in renal tumor angiogenesis and its expression may antagonize that of the PTEN tumor suppressor gene, affecting the Akt-survival pathway and promoting angiogenesis.

Isolation and characterization of human breast tumor-derived endothelial cells

Increasing evidence indicates that tumor-derived endothelial cells (TEC) possess a distinct and unique phenotype in respect to normal endothelial cells and may be able to acquire resistance to drugs. However, few functional studies are available on cultured TEC. In the present study, we obtained TEC from human breast carcinomas and, to dispel the possibility of contaminating tumor cells, we established six different clones that we characterized at a functional level. Breast TEC cell lines and clones did not undergo normal cell senescence in culture and showed constant expression of markers of endothelial activation and angiogenesis. These cells showed increased apoptosis resistance to vincristine and doxorubicin and increased random cell motility, as compared to normal micro-endothelial cells. In addition, breast TEC, at variance to normal endothelial cells, were able to grow and to organize in the absence of serum in capillary-like structures on Matrigel that persisted up to one week. These functional characteristics of breast TEC may be relevant for tumor angiogenesis and may indicate an increased pro-angiogenic activity of endothelial cells within the tumor. Moreover, our data suggest that TEC might be more appropriate for screening antiangiogenic drugs than normal endothelial cells.

Immortalized Mammary Epithelial Cells and Human Mammary Tumor-Derived Cells Display Increased Proliferation, Motility, and Invasiveness in Obese Rat Conditions Compared to Lean Rat Conditions in in Vitro Assays

Immortalized Mammary Epithelial Cells and Human Mammary Tumor-Derived Cells Display Increased Proliferation, Motility, and Invasiveness in Obese Rat Conditions Compared to Lean Rat Conditions in in Vitro Assays

Isolation and Functional Characterization of a Novel Organic Solute Carrier Protein, hOSCP1

We succeeded in isolating a novel organic solute carrier from a human placenta cDNA library. The isolated cDNA consisted of 1137 base pairs that encoded a 379-amino acid protein, hOSCP1. Northern blot and reverse transcription PCR analyses revealed that the hOSCP1 mRNA is expressed in the placenta and testis and weakly expressed in the thymus and small intestine. When expressed in Xenopus laevis oocytes, hOSCP1 mediated the high affinity transport of p-aminohippurate (PAH) (K(m) = 35.0 +/- 7.5 microm) and tetraethylammonium (K(m) = 62.3 +/- 12.2 microm) in a sodium-independent manner. However, the hOSCP1-expressing oocyte did not mediate the transport of L-carnitine. The transport of PAH by hOSCP1 was sensitive to pH, but the tetraethylammonium was not transported at the high pH examined. hOSCP1 transported prostaglandin E(2), prostaglandin F(2alpha), estrone sulfate, glutarate, L-leucine, L-ascorbic acid, and tetracycline. Thus, hOSCP1 also showed broad substrate specificity. A wide range of structurally unrelated organic compounds inhibited the hOSCP1-mediated PAH uptake. Immunohistochemical analysis revealed that the hOSCP1 protein is localized in the basal membrane of the syncytiotrophoblast in the human placenta. Our results suggest that hOSCP1 is a novel polyspecific organic solute carrier protein responsible for drug clearance from the human placenta.

Complementation of Human Papillomavirus Type 16 E6 and E7 by Jagged1-Specific Notch1-Phosphatidylinositol 3-Kinase Signaling Involves Pleiotropic Oncogenic Functions Independent of CBF1;Su(H);Lag-1 Activation

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.

Induction of Accelerated Senescence by γ Radiation in Human Solid Tumor-Derived Cell Lines Expressing Wild-Type TP53

Recent studies have demonstrated that p21WAF1 (now known as CDKN1A)-dependent and -independent accelerated senescence responses are a major determinant of the sensitivity of cancer cells to chemotherapeutic agents. The objective of the present study was to determine whether human solid tumor-derived cell lines that express wild-type TP53 can exhibit levels of CDKN1A induction after exposure to ionizing radiation that are sufficient to activate the accelerated senescence program. Exposure to 60Co gamma radiation (< or =8 Gy) triggered accelerated senescence in all five TP53 wild-type tumor cell lines examined, albeit to differing degrees. Three of the TP53 wild-type tumor cell lines, HCT116, A172 and SKNSH, activated the TP53 signaling pathway similarly to normal human fibroblasts, as judged by the nuclear accumulation of TP53, magnitude and duration of induction of CDKN1A mRNA and CDKN1A protein, and propensity to undergo accelerated senescence after radiation exposure. In the clonogenic survival assay, the degree of radiosensitivity of these three tumor cell lines was also in the range displayed by normal human fibroblasts. On the other hand, two other TP53 wild-type tumor cell lines, A498 and A375, did not maintain high levels of CDKN1A mRNA and CDKN1A protein at late times postirradiation and exhibited only low levels of accelerated senescence after radiation exposure. Studies with a CDKN1A knockout cell line (HCT116CDKN1A-/-) confirmed that the radiation-triggered accelerated senescence is dependent on CDKN1A function. We conclude that (1) clinically achievable doses of ionizing radiation can trigger CDKN1A-dependent accelerated senescence in some human tumor cell lines that express wild-type TP53; and (2) as previously documented for normal human fibroblasts, some TP53 wild-type tumor cell lines (e.g. HCT116, A172 and SKNSH) may lose their clonogenic potential in response to radiation-inflicted injury primarily through undergoing accelerated senescence.

Human Ovarian Tumor-Derived Chaperone-Rich Cell Lysates Activate Dentdritic Cells and Elicit T Cell Responses In Vitro

s for the 19th Annual Scientific Meeting of the International Society for Biological Therapy of Cancer, San Francisco, California, November 4-7, 2004: Dendritic Cells and Vaccines

A novel 4,4'-bispyridyl-5,5'-perfluoroalkyl-2,2'-bisoxazol with antitumoral activity via cell cycle arrest and induction of apoptosis.

The compound 6a is a novel bisoxazol derivative with high cytotoxic properties in vitro against different human tumor-derived cell lines and with similar efficiency against epithelial, haematopoietic and mesenchymal tumor cells. Although the molecular mechanism is not yet fully defined, cell cycle analysis revealed that 6a induces efficiently G0/G1 phase arrest in colon adenocarcinoma HT-29 cells in a dose- and time-dependent manner. Induction of cell death is observed, a possible explanation for the antiproliferative profile of the molecule. The compound was well tolerated at doses that allowed to examine its antitumor activity against human xenografts of the HT-29 cell line implanted s.c. in nude mice. Treatment of mice with 4 mg/kg of the compound resulted in a 60% inhibition of tumor growth. These observations support the use of 6a for the generation of more potent derivatives that could be used as new anticancer agents.

Choline kinase inhibition induces the increase in ceramides resulting in a highly specific and selective cytotoxic antitumoral strategy as a potential mechanism of action.

Choline kinase (ChoK, E.C. 2.7.1.32) is involved in the synthesis of phosphatidylcholine (PC), and has been found to be increased in human tumors and tumor-derived cell lines. Furthermore, ChoK inhibitors have been reported to show a potent and selective antitumoral activity both in vitro and in vivo. Here, we provide the basis for a rational understanding of the antitumoral activity of ChoK inhibitors. In normal cells, blockage of de novo phosphorylcholine (PCho) synthesis by inhibition of ChoK promotes the dephosphorylation of pRb, resulting in a reversible cell cycle arrest at G0/G1 phase. In contrast, ChoK inhibition in tumor cells renders cells unable to arrest in G0/G1 as manifested by a lack of pRb dephosphorylation. Furthermore, tumor cells specifically suffer a drastic wobble in the metabolism of main membrane lipids PC and sphingomyelin (SM). This lipid disruption results in the enlargement of the intracellular levels of ceramides. As a consequence, normal cells remain unaffected, but tumor cells are promoted to apoptosis. Thus, we provide in this study the rationale for the potential clinical use of ChoK inhibitors.

Roles of Stem Cell Factor/c-Kit and Effects of Glivec®/STI571 in Human Uveal Melanoma Cell Tumorigenesis

The B-Raf(V599E)-mediated constitutive activation of ERK1/2 is involved in establishing the transformed phenotype of some uveal melanoma cells (Calipel, A., Lefevre, G., Pouponnot, C., Mouriaux, F., Eychene, A., and Mascarelli, F. (2003) J. Biol. Chem. 278, 42409-42418). We have shown that stem cell factor (SCF) is involved in the proliferation of normal uveal melanocytes and that c-Kit is expressed in 75% of primary uveal melanomas. This suggests that the acquisition of autonomous growth during melanoma progression may involve the SCF/c-Kit axis. We used six human uveal melanoma tumor-derived cell lines and normal uveal melanocytes to characterize the SCF/c-Kit system and to assess its specific role in transformation. We investigated the possible roles of activating mutations in c-KIT, the overexpression of this gene, and ligand-dependent c-Kit overactivation in uveal melanoma cell tumorigenesis. Four cell lines (92.1, SP6.5, Mel270, and TP31) expressed both SCF and c-Kit, and none harbored the c-KIT mutations in exons 9, 11, 13, and 17 that have been shown to induce SCF-independent c-Kit activation. Melanoma cell proliferation was strongly inhibited by small interfering RNA-mediated depletion of c-Kit in these cells, despite the presence of (V599E)B-Raf in SP6.5 and TP31 cells. We characterized the signaling pathways involved in SCF/c-Kit-mediated cell growth and survival in normal and tumoral melanocytes and found that constitutive ERK1/2 activation played a key role in both the SCF/c-Kit autocrine loop and the gain of function of (V599E)B-Raf for melanoma cell proliferation and transformation. We also provide the first evidence that Glivec/STI571, a c-Kit tyrosine kinase inhibitor, could be used to treat uveal melanomas.

Exploratory Chemoinformatic Analysis of Cell Type-Selective Anticancer Drug Targeting

In pharmaceutical development, structure-activity relationship studies aim to identify characteristics of chemical structures associated with well-defined activity end points. While this goal-driven approach is ideally suited for lead development purposes, a more exploration-driven approach is needed to discover cell type-selective drug targeting mechanisms in complex data sets. Growth inhibition profiles across different cancer cell lines are potentially informative with respect to molecular mechanisms targeting the activity of anticancer agents to specific tumor cells, yet only a small number of mechanistic associations between chemical structure and growth inhibition profiles have been discovered to date. Here, we have applied an exhaustive statistical analysis strategy to more than 10000 compounds in the NCI's anticancer agent database to identify molecular substructures associated with specific cytotoxicity signatures against a panel of human tumor-derived cancer cell lines (the Developmental Therapeutics Program 60-cell line panel). Some of the most significant substructures conferring cell type-selective cytotoxic activity include a large family of delocalized lipophilic cations; chloropurines, chloropyrimidines, and thiazoles; organosulfur chelators and organometallic complexes; and an unexpectedly related family of alkyl-lysophospholipids and phosphate prodrugs. Information from cell-based assays and gene expression measurements have been related to substructures represented in the chemical space covered by the library, yielding several candidate targeting mechanisms.

Generation and characterization of a constitutively active Stat3 protein.

Stats are latent transcription factors involved in normal cellular signaling in response to cytokine or growth factor stimulation. Constitutive activation of Stats (primarily Stat3 and Stat5) has been implicated in growth dysregulation and oncogenesis. Furthermore, increased activation of Stats has been observed in several human tumors and tumor-derived cell lines. To assess the contribution of aberrant Stat activation in oncogenesis, we have created a chimeric molecule between Stat3beta and a portion of the Herpes simplex virus VP16 activation domain. The resulting protein, Stat3beta-VAD (VP16 activation domain), is tyrosine phosphorylated on Y705 and can bind DNA in the absence of upstream activation by c-Src or epidermal growth factor (EGF). Unlike Stat3alpha and Stat3beta, Stat3beta-VAD robustly activates transcription of several reporter genes without cytokine or growth factor stimulation. In addition, we show marked upregulation of the endogenous c-myc and c-fos genes upon inducible expression of Stat3beta-VAD in COS-7 cells. Our protein displays the constitutive transcriptional activation of Stat3alpha seen in human tumors and will be a valuable tool in screens for Stat3-regulated genes. In response to the established Stat3 involvement in human cancers, Stat3beta-VAD will also facilitate assessing the contribution of other cancer signaling cascades in the context of aberrant Stat3alpha activity in cancer development and progression.

Activity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells

The family of ubiquitin (Ub)-specific proteases (USP) removes Ub from Ub conjugates and regulates a variety of cellular processes. The human genome contains many putative USP-encoding genes, but little is known about USP tissue distribution, pattern of expression, activity, and substrate specificity. We have used a chemistry-based functional proteomics approach to identify active USPs in normal, virus-infected, and tumor-derived human cells. Depending on tissue origin and stage of activation/differentiation, different USP activity profiles were revealed. The activity of specific USPs, including USP5, -7, -9, -13, -15, and -22, was up-regulated by mitogen activation or virus infection in normal T and B lymphocytes. UCH-L1 was highly expressed in tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen-activated cells. Up-regulation of this USP was a late event in the establishment of Epstein-Barr virus-immortalized lymphoblastoid cell lines and correlated with enhanced proliferation, suggesting a possible role in growth transformation.

Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts

The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 106 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.

Expression regulation and genomic organization of human polynucleotide phosphorylase, hPNPaseold-35, a Type I interferon inducible early response gene

An overlapping pathway screening (OPS) approach designed to identify and clone genes displaying parallel expression profiles as a function of induction of terminal differentiation and cellular senescence in human cells identified a novel gene old-35. Sequence and functional analysis indicates that old-35 encodes human polynucleotide phosphorylase, hPNPase old-35 . Polynucleotide phosphorylases comprise a family of phosphate dependent 3′–5′ RNA exonucleases implicated in RNA regulation. Treatment of HO-1 human melanoma and additional diverse normal and tumor-derived human cell types with Type I interferon (IFN), IFN-β or IFN-α, induces hPNPase old-35 expression. To provide insights into the regulation of hPNPase old-35 , we cloned and analyzed the promoter region of this gene. These studies demonstrate that IFN-β controls hPNPase old-35 expression by transcriptional modulation rather than by altering mRNA stability. Transcriptional activation of hPNPase old-35 by IFN-β is primarily mediated by the interferon stimulatory response element (ISRE) present in its promoter. Analysis of hPNPase old-35 expression in cell lines defective in various IFN signaling molecules confirms that hPNPase old-35 expression is dependent upon the Janus activated kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Furthermore, gel shift analyses document that hPNPase old-35 is a direct target of the interferon stimulated gene factor 3 (ISGF3) complex. The hPNPase old-35 gene spans ∼54 kb of genomic DNA and is distributed on 28 exons and 27 introns. hPNPase old-35 maps to 2p15–2p16.1, a region implicated in hereditary nonpolyposis colorectal cancer, Carney complex, Doyne′s honeycomb retinal dystrophy and several other diseases. To provide insights into PNPase function in vivo, we have also cloned the mouse PNPase old-35 cDNA, mPNPase old-35 . Induction of hPNPase old-35 by IFN treatment as well as during differentiation and senescence suggest that this gene may play a significant role in regulating cellular growth and that overlapping gene expression changes, also induced by IFN, may contribute to these important physiological processes.