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Oncoproteins encoded by dominant oncogenes have long been considered as targets for chemotherapeutic intervention. However, oncogenic transcription factors have often been dismissed as "undruggable." Members of the Myc family of transcription factors have been identified as promising targets for cancer chemotherapy in multiple publications reporting the requirement of Myc proteins for maintenance of almost every type of tumor. Here, we describe cell-based approaches to identify c-Myc small molecule inhibitors by screening complex libraries of diverse small molecules based on Myc functionality and specificity.

The transcriptional co-regulators YAP and TAZ pair primarily with the TEAD family of transcription factors to elicit a gene expression signature that plays a prominent role in cancer development, progression and metastasis. YAP and TAZ endow cells with various oncogenic traits such that they sustain proliferation, inhibit apoptosis, maintain stemness, respond to mechanical stimuli, engineer metabolism, promote angiogenesis, suppress immune response and develop resistance to therapies. Therefore, inhibiting YAP/TAZ- TEAD is an attractive and viable option for novel cancer therapy. It is exciting to know that many drugs already in the clinic restrict YAP/TAZ activities and several novel YAP/TAZ inhibitors are currently under development. We have classified YAP/TAZ-inhibiting drugs into three groups. Group I drugs act on the upstream regulators that are stimulators of YAP/TAZ activities. Many of the Group I drugs have the potential to be repurposed as YAP/TAZ indirect inhibitors to treat various solid cancers. Group II modalities act directly on YAP/TAZ or TEADs and disrupt their interaction; targeting TEADs has emerged as a novel option to inhibit YAP/TAZ, as TEADs are major mediators of their oncogenic programs. TEADs can also be leveraged on using small molecules to activate YAP/TAZ-dependent gene expression for use in regenerative medicine. Group III drugs focus on targeting one of the oncogenic downstream YAP/TAZ transcriptional target genes. With the right strategy and impetus, it is not far-fetched to expect a repurposed group I drug or a novel group II drug to combat YAP and TAZ in cancers in the near future., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The era of personalized medicine for advanced-stage non-small-cell lung cancer (NSCLC) began when biomarker-based evidence of molecular pathway and/or oncogene addiction of the tumour became mandatory for the allocation of specific targeted therapies. More recently, the immunotherapy revolution, specifically, the development of immune-checkpoint inhibitors (ICIs), has dramatically altered the NSCLC treatment landscape. Herein, we compare and contrast the clinical development of immunotherapy and oncogene-directed therapy for NSCLC, focusing on the role of predictive biomarkers. Immunotherapy biomarkers are fundamentally different from oncogene biomarkers in that they are continuous rather than categorical (binary), spatially and temporally variable and reliant on multiple complex interactions rather than a single, dominant determinant. The performance of predictive biomarkers for ICIs might be improved by combining different markers to reduce the assumptive risks associated with each one. Novel combinations with chemotherapy and ICIs complicate biomarker discovery but do not decrease the value of the markers identified. Perfectly predictive biomarkers of benefit from immunotherapy are unlikely to be identified, although exclusionary biomarkers of minimal benefit or an unacceptable risk of toxicity might be feasible. The clinical adoption and applicability of such biomarkers might vary depending on line of treatment, the available therapeutic alternatives and health economic considerations.

Immunotherapy is being tested intensively in clinical trials for prostate cancer; it includes immune checkpoint inhibition, prostate specific antigen (PSA) vaccines and dendritic cell-based strategies. Despite increasing evidence for clinical responses, the consensus of multiple trials is that prostate cancers are poorly responsive to immunotherapy. Prostate cancer has a high degree of pathological and genetic heterogeneity compared to other cancer types, which may account for immunotherapeutic resistance. This hypothesis also implies that select types of prostate tumors may be differentially responsive to immune-based strategies and that the clinical stage, pathological grade and underlying genetic landscape may be important criteria in identifying tumors that respond to immune therapies. One strategy is to target oncogenic driver pathways in combination with immunotherapies with the goal of overcoming tumor immunity and broadening the number of patients achieving a clinical response. In this analysis, we address the hypothesis that driver oncogenic signaling pathways regulate cancer progression, tumor immunity and resistance to current immune therapeutics in prostate cancer. We propose that increased responsiveness may be achieved through the combined use of immunotherapies and inhibitors targeting tumor cell autonomous pathways that contribute towards anti-tumor immunity in patients with prostate cancer., Competing Interests: The authors indicate no potential conflicts of interest.

Immunomodulatory drugs (IMiDs), such as thalidomide and its derivatives lenalidomide and pomalidomide, are key treatment modalities for hematologic malignancies, particularly multiple myeloma (MM) and del(5q) myelodysplastic syndrome (MDS). Cereblon (CRBN), a substrate receptor of the CRL4 ubiquitin ligase complex, is the primary target by which IMiDs mediate anticancer and teratogenic effects. Here we identify a ubiquitin-independent physiological chaperone-like function of CRBN that promotes maturation of the basigin (BSG; also known as CD147) and solute carrier family 16 member 1 (SLC16A1; also known as MCT1) proteins. This process allows for the formation and activation of the CD147-MCT1 transmembrane complex, which promotes various biological functions, including angiogenesis, proliferation, invasion and lactate export. We found that IMiDs outcompete CRBN for binding to CD147 and MCT1, leading to destabilization of the CD147-MCT1 complex. Accordingly, IMiD-sensitive MM cells lose CD147 and MCT1 expression after being exposed to IMiDs, whereas IMiD-resistant cells retain their expression. Furthermore, del(5q) MDS cells have elevated CD147 expression, which is attenuated after IMiD treatment. Finally, we show that BSG (CD147) knockdown phenocopies the teratogenic effects of thalidomide exposure in zebrafish. These findings provide a common mechanistic framework to explain both the teratogenic and pleiotropic antitumor effects of IMiDs.

Gene transcription analysis in clinical tumor samples can help with diagnosis, prognosis, and treatment of cancers. We aimed to identify the optimal reference genes for reliable expression analysis in various tumor samples by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Using a one-step TaqMan-based qRT-PCR, 5 commonly used reference genes (ACTB, GAPDH, RPLPO, GUSB, and TFRC) and 10 anticancer drug-related genes (TYMS, RRM1, TUBB3, STMN1, TOP2A, EGFR, VEGFR2, HER2, ERCC1, and BRCA1) were analyzed in 327 tissue samples from lung, rectal, colon, gastric, esophageal, and breast tumors. According to the expression stability assessments obtained by using three programs (geNorm, NormFinder, and BestKeeper) and a comprehensive ranking method, the optimal reference genes for lung, gastric, esophageal, and breast tumors were RPLPO, GAPDH, ACTB, and ACTB, respectively. For rectal tumors, a combination of the 3 most stable genes (GUSB, ACTB, and RPLPO) was suitable for qRT-PCR, whereas for colon tumors, a combination of the 4 most stable genes (GAPDH, ACTB, GUSB, and RPLPO) was optimal for qRT-PCR. Based on the expression data of target genes normalized against selected reference genes, the principal component analysis revealed 4 expression patterns in 6 different tissues. One pattern was observed in gastric, rectal, and colon tumor tissues, which are gastrointestinal tumors. Expressions in the breast, lung, and esophageal tissues were separately represented as one pattern. Our results could facilitate the practice of personalized cancer medicine based on the gene expression profile of the patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Nucleophosmin (NPM1) mutations represent an attractive therapeutic target in acute myeloid leukemia (AML) because they are common (∼30% AML), stable, and behave as a founder genetic lesion. Oncoprotein targeting can be a successful strategy to treat AML, as proved in acute promyelocytic leukemia by treatment with all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO), which degrade the promyelocytic leukemia (PML)-retinoic acid receptor fusion protein. Adjunct of ATRA to chemotherapy was reported to be beneficial for NPM1-mutated AML patients. Leukemic cells with NPM1 mutation also showed sensibility to ATO in vitro. Here, we explore the mechanisms underlying these observations and show that ATO/ATRA induce proteasome-dependent degradation of NPM1 leukemic protein and apoptosis in NPM1-mutated AML cell lines and primary patients' cells. We also show that PML intracellular distribution is altered in NPM1-mutated AML cells and reverted by arsenic through oxidative stress induction. Interestingly, similarly to what was described for PML, oxidative stress also mediates ATO-induced degradation of the NPM1 mutant oncoprotein. Strikingly, NPM1 mutant downregulation by ATO/ATRA was shown to potentiate response to the anthracyclin daunorubicin. These findings provide experimental evidence for further exploring ATO/ATRA in preclinical NPM1-mutated AML in vivo models and a rationale for exploiting these compounds in chemotherapeutic regimens in clinics., (© 2015 by The American Society of Hematology.)

Lutein is a carotenoid pigment present in fruits and vegetables that has anti-inflammatory and antitumor properties. In this study, we examined the effect of lutein on proliferation and survival-associated genes in prostate cancer (PC-3) cells. We found that in vitro culture of PC-3 cells with lutein induced mild decrease in proliferation that improved in combination treatment with peroxisome proliferator-activated receptor gamma (PPARγ) agonists and other chemotherapeutic agents. Flow cytometry analyses showed that lutein improved drug-induced cell cycle arrest and apoptosis in prostate cancer. Gene array and quantitative reverse transcription-polymerase chain reaction analyses showed that lutein altered the expression of growth and apoptosis-associated biomarker genes in PC-3 cells. These findings highlight that lutein modulates the expression of growth and survival-associated genes in prostate cancer cells.

Background and Objective: Sonic hedgehog protein (SHH) is a mitogen that stimulates cell proliferation. Cyclosporine A enhances the proliferation of gingival cells; however, the relationships of SHH to cyclosporine A or to cyclosporine A-enhanced gingival cell proliferation have not been described., Material and Methods: Here, we investigated SHH expression in gingiva in vitro and in vivo after cyclosporine A treatment and tested the effect of SHH inhibition on cyclosporine A-enhanced gingival fibroblast proliferation in vitro., Results: In human gingival fibroblasts, cyclosporine A treatment increased the expression of SHH transcripts and SHH protein, and stimulated cell proliferation; the addition of cyclopamine, an SHH signaling inhibitor, suppressed cyclosporine A-enhanced cell proliferation. Up-regulated expression of SHH and up-regulation of proliferating cell nuclear antigen transcripts and protein were observed in the edentulous gingiva of cyclosporine A-treated rats., Conclusion: Cyclosporine A up-regulates gingival SHH expression in vitro and in vivo, and the inhibition of the SHH pathway counteracts the stimulatory effect of cyclosporine A on gingival fibroblast proliferation. Therefore, we suggest that SHH mediates a novel molecular mechanism for cyclosporine A-induced gingival complications., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The remarkable success of epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors in patients with EGFR mutations and ALK rearrangements, respectively, introduced the era of targeted therapy in advanced non-small cell lung cancer (NSCLC), shifting treatment from platinum-based combination chemotherapy to molecularly tailored therapy. Recent genomic studies in lung adenocarcinoma identified other potential therapeutic targets, including ROS1 rearrangements, RET fusions, MET amplification, and activating mutations in BRAF, HER2, and KRAS in frequencies exceeding 1%. Lung cancers that harbor these genomic changes can potentially be targeted with agents approved for other indications or under clinical development. The need to generate increasing amounts of genomic information should prompt health-care providers to be mindful of the amounts of tissue needed for these assays when planning diagnostic procedures. In this review, we summarize oncogenic drivers in NSCLC that can be currently detected, highlight their potential therapeutic implications, and discuss practical considerations for successful application of tumor genotyping in clinical decision making.

Purpose: To investigate the effects of (-)-epigallocatechin-3-gallate (EGCG) on human papillomavirus (HPV)-16 oncoprotein-induced angiogenesis in non-small cell lung cancer (NSCLC) cells and the underlying mechanisms., Methods: NSCLC cells (A549 and NCI-H460) transfected with EGFP plasmids containing HPV-16 E6 or E7 oncogene were treated with different concentrations of EGCG for 16 h. The effects of EGCG on angiogenesis in vitro and in vivo were observed. The expression of HIF-1α, p-Akt, and p-ERK1/2 proteins in NSCLC cells was analyzed by Western blot. The levels of HIF-1α mRNA in NSCLC cells were detected by real-time RT-PCR. The concentration of VEGF and IL-8 in the conditioned media was determined by ELISA. HIF-1α, VEGF, and CD31 expression in A549 xenografted tumors of nude mice was analyzed by immunohistochemistry., Results: HPV-16 E6 and E7 oncoproteins HIF-1α-dependently promoted angiogenesis in vitro and in vivo, which was inhibited by EGCG. Mechanistically, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression in NSCLC cells. Additionally, 50 and 100 μmol/L of EGCG significantly reduced the secretion of VEGF and IL-8 proteins induced by HPV-16 E7 oncoprotein in NSCLC A549 cells. Meanwhile, HPV-16 E6 and E7 oncoproteins HIF-1α-dependently enhanced Akt activation in A549 cells, which was suppressed by EGCG. Furthermore, EGCG inhibited HPV-16 oncoprotein-induced HIF-1α and HIF-1α-dependent VEGF and CD31 expression in A549 xenografted tumors., Conclusions: EGCG inhibited HPV-16 oncoprotein-induced angiogenesis conferred by NSCLC through the inhibition of HIF-1α protein expression and HIF-1α-dependent expression of VEGF, IL-8, and CD31 as well as activation of Akt, suggesting that HIF-1α may be a potential target of EGCG against HPV-related NSCLC angiogenesis.

Background: To clarify the efficacy of grape seed procyanidin (GSP) on antiproliferative effects related to p53 functional status of oral squamous cell carcinoma (OSCC) for its chemoadjuvant potential., Methods: We used GSP to investigate SCC-25 cells with wild-type p53 gene and OEC-M1 cells with mutant p53 gene for the assessment of antiproliferative effects including cell viability, cell cycle, apoptosis, migration and invasion potential, and alterations of associated oncoproteins involved in cellular and molecular events., Results: The findings suggest that GSP on OEC-M1 cells leads to cell cycle arrest by increasing the expression of p21(Cip1) /p27(Kip1) protein without functioning mitochondria-mediated apoptosis, whereas GSP on SCC-25 cells inhibits cell proliferation via both G1-phase arrest and mitochondria-mediated apoptosis in a dose-dependent manner as a result of alterations of Bcl-2. GSP also inhibits the migration and invasion of both cells, which are associated with the suppression of matrix metalloproteinases (MMPs), MMP-2 and MMP-9., Conclusion: Antiproliferative effectiveness of GSP is closely associated with the p53 status of OSCC cells. GSP displays chemoadjuvant potential via cell cycle blockage and apoptotic induction. Our findings clearly suggest that GSP may play a role as a novel chemopreventive or therapeutic agent for OSCC., (© 2011 John Wiley & Sons A/S.)

The D(2) dopamine receptor (D(2)R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D(2)R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D(2)R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D(2)R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D(2)Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D(2)(+/-) mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D(2)R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D(2)R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D(2)R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D(2)R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.

Parkinson disease is caused by the death of midbrain dopamine neurons from oxidative stress, abnormal protein aggregation, and genetic predisposition. In 2003, Bonifati et al. (23) found that a single amino acid mutation in the DJ-1 protein was associated with early-onset, autosomal recessive Parkinson disease (PARK7). The mutation L166P prevents dimerization that is essential for the antioxidant and gene regulatory activity of the DJ-1 protein. Because low levels of DJ-1 cause Parkinson, we reasoned that overexpression might stop the disease. We found that overexpression of DJ-1 improved tolerance to oxidative stress by selectively up-regulating the rate-limiting step in glutathione synthesis. When we imposed a different metabolic insult, A53T mutant α-synuclein, we found that DJ-1 turned on production of the chaperone protein Hsp-70 without affecting glutathione synthesis. After screening a number of small molecules, we have found that the histone deacetylase inhibitor phenylbutyrate increases DJ-1 expression by 300% in the N27 dopamine cell line and rescues cells from oxidative stress and mutant α-synuclein toxicity. In mice, phenylbutyrate treatment leads to a 260% increase in brain DJ-1 levels and protects dopamine neurons against 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) toxicity. In a transgenic mouse model of diffuse Lewy body disease, long-term administration of phenylbutyrate reduces α-synuclein aggregation in brain and prevents age-related deterioration in motor and cognitive function. We conclude that drugs that up-regulate DJ-1 gene expression may slow the progression of Parkinson disease by moderating oxidative stress and protein aggregation.

A number of polyamine analogs are currently used in various clinical trials as cancer treatment and it is important to investigate their effects not only on cancer cells but also on normal cells. Treatment with polyamine analogs depletes cells of polyamines and inhibits cell proliferation, but the analogs cannot take over the normal function of the natural polyamines in the cell. In this study, the normal-like breast epithelial cell line MCF-10A was treated with the polyamine analog N',N"-diethylnorspermine (DENSPM). The cells were then studied using a bromodeoxyuridine- DNA flow cytometry method as well as western blot. The ability of both normal-like and breast cancer cells to recover from DENSPM treatment was also studied. DENSPM treatment of MCF-10A cells resulted in a prolongation of the S and G2 +M phases, followed by a G1/S block. The p53/p21/RB1 pathway was involved in the G1/S block as shown by increased levels of p53 and p21 detected by western blot. Decreased levels of cyclin E1, cyclin A2, and cyclin B1 in DENSPM-treated cells can explain the prolongation of cell cycle phases that occurred before the G1/S block. We also show that MCF-10A cells rapidly recover from DENSPM-induced growth inhibition in contrast to four human breast cancer cell lines. The goal of cancer treatment is to cause minimal and reversible damage to normal cells, while cancer cells should be eliminated. Altogether, the data show that treatment with polyamine analogs spares normal cells, while negatively affecting the cancer cells.

Although a loss-of-function type mutation was identified in familial Parkinson's disease PARK7, the wild-type of DJ-1 is known to act as an oxidative stress sensor in neuronal cells. Recently, we found a DJ-1 modulator UCP0054278 by in silico virtual screening. In this study, we determined the neuroprotective effects of UCP0054278 against focal ischemia-induced neurodegeneration in rats. Hydrogen peroxide-induced cell death and the production of reactive oxygen species were significantly inhibited by UCP0054278 in normal SH-SY5Y cells, but not in DJ-1-knockdown cells. These results suggest that UCP0054278 interacts with endogenous DJ-1 and then exhibits antioxidant and neuroprotective responses.

Background: Use of retinoic acid (RA) has become the standard of care in the treatment of high risk neuroblastoma (NB). In vitro, RA induces growth arrest and differentiation, an effect that likely underlies its activity in the clinical setting. An important event in differentiation is the transcriptional downregulation of the MYCN oncogene, which is frequently activated in aggressive tumors. While it is known that Sp1/Sp3 and E2F are necessary to drive basal MYCN expression, the mechanism for its downregulation by RA remains enigmatic. Changes in E2F binding have been reported, however these occurred after the actual transcriptional response. Here, post-translational modifications of Sp proteins were examined as an alternate mechanism of RA-mediated promoter regulation., Procedure: Western blot was used to evaluate steady state levels of nuclear/cytoplasmic Sp1/Sp3. Promoter binding and DNA conformation were determined by gel shift, circular permutation, and chromatin immunoprecipitation assays. Immunoprecipitation/western and (32)P-phosphoamino analyses were used to detect glycosylation, acetylation, sumoylation, and phosphorylation., Results: RA did not affect the cellular level of Sp1/Sp3 proteins, their nuclear/cytoplasmic distribution, ability to bind the MYCN promoter, degree of Sp-induced DNA bending, or post-translational modifications., Conclusions: MYCN RA response is not mediated solely though the region controlling basal activity. RA may be exerting its effects via multiple non-adjacent regulatory regions, potentially including basal motifs, either within the MYCN promoter or distally, on the same or even different chromosomes. Such cooperative trans-type DNA-protein interactions could explain the inaccessibility of this mechanism to the locus-specific approaches employed up to this point., ((c) 2008 Wiley-Liss, Inc.)

Background: Mechanisms regulating the transition from hormone responsive to hormone refractory prostate cancer (PCa) have remained unclear., Methods: We analyzed androgen and anti-androgen treatment on endogenous AR activity in primary human prostate epithelial (HPE) cells cultured directly from patient radical prostatectomy specimens utilizing a transiently infected gene reporter (TIGR) assay., Results: Flutamide treatment exhibited agonist activities in HPE cells derived from tumor and non-tumor specimens which contained wild-type AR. After proteomic comparison of these cells to those where flutamide functioned normally as an antagonist, we identified DJ-1, a positive regulator of AR. DJ-1 expression increased in HPE and LNCaP cells during flutamide treatment as a result of DJ-1 protein stabilization., Conclusion: Stabilization of AR and its co-regulators in the absence of androgen may partially account for anti-androgen withdrawal syndrome and potentially contribute to the development of hormone refractory PCa., ((c) 2006 Wiley-Liss, Inc.)

Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of the flanking "extein" sequences. Previously we reported the directed evolution of an intein-based molecular switch in which intein splicing in yeast cells was made dependent on the cell-permeable small molecule 4-hydroxytamoxifen (4-HT). Here we show that these evolved inteins are effective means of rendering protein function and biological signaling pathway activation dependent on 4-HT in mammalian cells. We have characterized the generality, speed, and dose dependence of ligand-induced protein splicing in murine NIH3T3 cells and in human HEK293 cells. Evolved inteins were used to control in mammalian cells the function of Gli1 and a truncated form of Gli3, two transcriptional mediators of the Hedgehog signaling pathway. Finally, we show that a complex biological process such as osteoblast differentiation can be made dependent on 4-HT using the evolved intein system. Our findings suggest that evolved small-molecule-dependent inteins may serve as a general means of achieving gene-specific, dose-dependent, post-translational, and small-molecule-induced control over protein activity in mammalian systems.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause a multitude of detrimental effects to developing zebrafish (Danio rerio). Previously, we demonstrated that jaw growth was impaired by TCDD exposure, but the exact mechanism underlying these malformations remained unknown. In the present study, we investigated the involvement of hedgehog genes and their downstream signaling in TCDD-mediated jaw malformation. We demonstrate that the developing lower jaw expresses sonic hedgehog a (shha), sonic hedgehog b (shhb) and their receptors, patched1 (ptc1) and patched2 (ptc2), as well as the downstream transcription factors, gli1 and gli2a. Loss of Hh signaling in mutants (sonic you) and larvae treated with a Hh inhibitor (cyclopamine), resulted in similar effects as those caused by TCDD. Moreover, TCDD exposure caused downregulation of shha and shhb in a manner dependent on aryl hydrocarbon receptor 2 (ahr2). Although this suggested an involvement of Hh signaling in TCDD-mediated impairment of jaw growth, we did not observe downregulation of ptc1 and ptc2, receptors dependent on Hh signaling. Furthermore, while the overall occurrence of apoptosis in the developing jaw was minimal, it was significantly increased in larvae treated with cyclopamine. In contrast, both TCDD and cyclopamine markedly reduced immunoreactivity against phosphorylated histone 3, a cell proliferation marker in the developing jaw. Taken together, our data suggest that Ahr2-mediated downregulation of Hh signaling, leading to a failure of cell proliferation, contributes to TCDD induced inhibition of lower jaw growth. TCDD may impair jaw growth through other pathway(s) in addition to Hh signaling.

Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.

Gastric adenocarcinoma (GA) is a significant cause of mortality worldwide. The molecular mechanisms of GA remain poorly characterised. Our aim was to characterise the functional activity of the computationally identified genes, NET 1 and MYEOV in GA. Digital Differential Display was used to identify genes altered expression in GA-derived EST libraries. mRNA levels of a subset of genes were quantitated by qPCR in a panel of cell lines and tumour tissue. The effect of pro- and anti-inflammatory stimuli on gene expression was investigated. Cell proliferation and invasion were measured using in an in-vitro GA model following inhibition of expression using siRNA. In all, 23 genes not previously reported in association with GA were identified. Two genes, Net1 and Myeov, were selected for further analysis and increased expression was detected in GA tissue compared to paired normal tissue using quantitative PCR. siRNA-mediated downregulation of Net1 and Myeov resulted in decreased proliferation and invasion of gastric cancer cells in vitro. These functional studies highlight a putative role for NET1 and Myeov in the development and progression of gastric cancer. These genes may provide important targets for intervention in GA, evidenced by their role in promoting invasion and proliferation, key phenotypic hallmarks of cancer cells.

Protein kinase Balpha (PKBalpha) is a key regulator of metabolism, proliferation and differentiation. We have explored the role of PKBalpha in adipogenesis using wild-type and PKBalpha-knockout mouse embryonic fibroblasts (MEFs) and show that lack of PKBalpha prevents MEF differentiation into adipocytes. Expression of ectopic PKBalpha in PKBalpha-deficient cells restores adipogenesis. We identified 80 genes whose expression was upregulated in wild-type MEFs during adipogenesis but whose expression was significantly reduced in PKBalpha-deficient MEFs under the same conditions. Significantly, the regulator of adipogenesis Krüppel-like transcription factor 15 gene expression was downregulated in PKBalpha-deficient MEFs but could be restored by expressing an active PKBalpha in the deficient cells. The level of lipocalin 2, renin 1 and receptor-activity-modifying protein 3 genes expressed by adipose cells was also decreased in PKBalpha-deficient MEFs, and are inhibited by LY294002 treatment during early adipocyte differentiation of 3T3-L1 cells. The results underscore an essential role for PKBalpha in the transcriptional program required for adipogenesis.

We have investigated the effect of HER-2 overexpression on resistance to the aromatase inhibitor letrozole in MCF-7 breast cancer cells stably expressing cellular aromatase (MCF-7/CA). MCF-7/CA cells overexpressing HER-2 showed a >2-fold increase in estrogen receptor (ER)-mediated transcriptional reporter activity upon treatment with androstenedione compared with vector-only control MCF-7/CA cells. Co-treatment with letrozole did not abrogate androstenedione-induced transcription and cell proliferation in HER-2-overexpressing cells. Chromatin immunoprecipitation assays using cross-linked protein-DNA from MCF-7/CA/HER-2 cells indicated ligand-independent association of the ERalpha coactivators AIB-1 and CBP to the promoter region of the estrogen-responsive pS2 gene. Upon treatment with androstenedione, there were increased associations of AIB1 and CBP with the pS2 promoter in the HER-2-overexpressing compared with control MCF-7/CA cells. These results suggest that ligand-independent recruitment of coactivator complexes to estrogen-responsive promoters as a result of HER-2 overexpression may play a role in the development of letrozole resistance.

Steroid receptor coactivator-3 (SRC-3/AIB1) is an oncogene frequently amplified and overexpressed in breast cancers. Here we report that SRC-3 interacts with REGgamma, a proteasome activator known to stimulate the trypsin-like activity of the 20S proteasome. RNAi knockdown and gain-of-function experiments suggest that REGgamma promotes SRC-3 protein degradation. Cellular levels of REGgamma expression affect estrogen-receptor target-gene expression and cell growth as a result of its ability to promote degradation of the SRC-3 protein. In vitro proteasome proteolysis assays using purified REGgamma, SRC-3, and the 20S proteasome reinforce these conclusions and demonstrate that REGgamma promotes the degradation of SRC-3 in a ubiquitin- and ATP-independent manner. This work demonstrates the first example of a physiologically relevant endogenous cellular target for the REGgamma-proteasome complex. It also highlights the fact that an alternative mode of proteasome-mediated protein degradation, independent of the 19S proteasome regulatory cap, targets the SRC-3 protein for degradation.

Background: Non-steroidal anti-inflammatory drugs such as indomethacin can inhibit the growth of tumors through both the cyclooxygenase-2 (COX-2) dependant and COX-2 independent pathways, but the exact mechanism has not yet been shown. In our previous study, COX-2 independent proteins (Bfl-1, WISP-1 and proliferating cell nuclear antigens [PCNA]) in indomethacin-treated colorectal cancer cells with the use of proteomics technology had been identified., Objectives: To study and confirm the effect of indomethacin on the expression of Bfl-1, WISP-1 and PCNA in human colon cancer line HCT116 cells and the COX-2 independent tumor inhibiting pathway., Methods: Human colon cancer cell line HCT116 cells were divided into a treatment with indomethacin (IC 50) group, and a treatment with dimethyl sulfoxide (DMSO) as a control group for 48 h. The expression of Bfl-1, WISP-1 and PCNA, mRNA and protein were determined by a real-time quantitative PCR and Western blot, respectively., Results: Indomethacin down-regulated the expression of Bfl-1, WISP-1 and PCNA mRNA in vitro (9.53 +/- 0.15 vs 27.87 +/- 0.12, 7.37 +/- 0.58 vs 20.17 +/- 0.58, 5.17 +/- 0.06 vs 0.87 +/- 0.06). Indomethacin also down-regulated the expression of Bfl-1, WISP-1 and PCNA protein (40.01 +/- 1.61 vs 43.76 +/- 1.63, 22.50 +/- 1.17 vs 30.30 +/- 1.55, 17.69 +/- 1.18 vs 20.80 +/- 1.08)., Conclusions: Inducing apoptosis and inhibiting proliferation contribute to the anticancer activity of indomethacin via COX-2 independent pathway of Bfl-1, WISP-1 and PCNA. This further confirms the results of our previous study.

The development of new cancer treatments is quickly evolving away from traditional practices of the last 25 years. This change is occurring not only at the technical level, but also conceptually as the human genome is unravelled and decades of research contribute to our understanding of the molecular complexity of this disease. It is anticipated that disease initiation and progression is dictated by an understandable set of acquired capabilities. Knowledge of the molecular events associated with these acquired capabilities will allow the development of targeted agents coupled with new biomarkers for the prevention of cancer progression. This will have a profound influence on how drugs are developed, approved, and used by the medical community. The Food and Drug Administration (FDA) has over 400 Investigational New Drug (IND) applications for cancer in its portfolio, which increasingly involve molecular targets and genomic applications. However, only one-fifth of IND agents succeed in New Drug Application (NDA) and there is more expense and uncertainty around successful drug development than ever before. Biomarkers should help the success rate of INDs by enhancing the link between target and disease as well as in improving patient selection and monitoring response. In this review, we discuss how biomarkers can be used for target validation and pharmacodynamic modeling in preclinical drug discovery. We then explore the use of biomarkers in clinical development from proof of mechanism to proof of concept studies, as well as their use in the prevention setting.

Farnesyltransferase (FT) inhibitors were originally designed as anticancer agents, and were thought to act by inhibiting the farnesylation of mutant Ras proteins. However, these compounds were subsequently demonstrated to have antitumor effects even in the absence of Ras mutations and it has now become clear that other protein targets are involved. This article discusses the preclinical and clinical development of FT inhibitors. To date, tipifarnib (Zarnestra; Janssen Pharmaceutica NV) and lonafarnib (Sarasar; Schering-Plough Research Institute) are the only two FT inhibitors to have been evaluated in phase III clinical trials. The clinical results of these two compounds are presented below, with emphasis on ways of enhancing the possibility of a successful FT inhibitor anticancer drug. Details of new FT inhibitors disclosed since the beginning of 2003 are also included.

Ret oncoproteins expressed in thyroid carcinomas represent possible targets for therapeutic intervention. Oncogenic activation of the receptor tyrosine kinase encoding RET gene occurs typically by gene rearrangement in papillary thyroid carcinomas (PTC) and by missense mutation in medullary thyroid carcinomas (MTC). These genetic alterations lead to the expression of deregulated products characterized by ligand-independent activation of the intrinsic tyrosine kinase of Ret. Such features suggest the possibility of using specific tyrosine kinase inhibitors to block the Ret oncoproteins signaling. The present report summarizes the cellular effects of the arylidene 2-indolinone Ret inhibitor RPI-1 (formerly Cpd1) on the human PTC cell line TPC-1 which spontaneously harbors the RET/PTC1 oncogene. The results provide evidence that RPI-1 is able to inhibit cell growth and to interfere with Ret/ptc1-driven signaling. These findings support a role for Ret oncoproteins as therapeutic targets and the pharmacological interest of RPI-1 as a candidate drug for preclinical evaluation on thyroid tumors expressing RET oncogenes.

Medullary thyroid carcinoma (MTC) responds very poorly to chemotherapy. Mutations in the RET gene are critical for MTC pathogenesis. RET therefore represents a rational target for the development of novel MTC therapies. The accumulation of evidence from laboratory studies strongly suggests that PP1 inhibitor is a cytostatic agent for cells expressing RET oncoproteins. PP1 functions as a potent and selective inhibitor of RET oncoprotein phosphorylation, promoting its proteasomal degradation.

Background: The RET gene encodes a tyrosine kinase receptor involved in different human neurocristopathies, such as specific neuroendocrine tumours and Hirschsprung disease (HSCR). Gene expression is developmentally regulated and the RET transcript is undetectable in most adult cells, including lymphocytes. The impossibility of performing functional studies on RET mRNA has to date limited the detection and characterisation of an indefinite proportion of gene anomalies that cannot be identified by conventional DNA genomic screening in HSCR cases., Aims: Development of a protocol suitable to activate RET expression in RET negative cell lines and therefore to investigate directly RET mRNA, extending the conventional gene mutation analysis to detection of splicing anomalies and impaired expression of the RET gene., Methods: The effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on rescuing RET expression was tested by one round of reverse transcription- polymerase chain reaction from total RNA of treated lymphoblasts from both HSCR patients and control individuals., Results: Analysis of RET expression was possible by NaB treatment of RET negative cells, such as lymphoblasts. This treatment allowed us to detect impaired RET expression as well as a splicing defect in two HSCR patients previously believed to be devoid of any gene abnormality., Conclusions: The full application of the proposed protocol in most of the unexplained HSCR cases will allow us to establish the precise role of RET not only in causing but also in predisposing to HSCR pathogenesis.

We used synthetic lethal high-throughput screening to interrogate 23,550 compounds for their ability to kill engineered tumorigenic cells but not their isogenic normal cell counterparts. We identified known and novel compounds with genotype-selective activity, including doxorubicin, daunorubicin, mitoxantrone, camptothecin, sangivamycin, echinomycin, bouvardin, NSC146109, and a novel compound that we named erastin. These compounds have increased activity in the presence of hTERT, the SV40 large and small T oncoproteins, the human papillomavirus type 16 (HPV) E6 and E7 oncoproteins, and oncogenic HRAS. We found that overexpressing hTERT and either E7 or LT increased expression of topoisomerase 2alpha and that overexpressing RAS(V12) and ST both increased expression of topoisomerase 1 and sensitized cells to a nonapoptotic cell death process initiated by erastin.

The mitogen-activated protein kinases (MAPKs) are a group of signal transducers with oncogenic potential in an assortment of cell types. Dysregulated signalling from any of the members of this family has been shown to result in development of human malignancies. Consequently, the collective goal of the scientific community is to inhibit aberrant signalling initiated from these molecules whilst minimising toxicity associated with such inhibition. This review covers events responsible for MAPK activation in detail, with an emphasis placed upon possible points of pharmacological intervention. A discussion addressing numerous chemotherapeutic approaches that have been developed over the previous decade for MAPK inhibition is also included. In addition, emphasis is placed upon the various arrays of kinase inhibitors, small molecule inhibitors, competitive inhibitors, nucleic acid aptamers and other molecules which have been proven effective in prevention of MAPK signalling. Finally, the potential therapeutic promise of many of these compounds is addressed in a manner that encompasses the complexities of MAPK signal transduction, in addition to concerns surrounding the development of drug resistance.

The AML1 (RUNX1)-MTG8 (ETO) fusion transcription factor generated by the t(8;21) translocation is believed to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia. To elucidate the role of AML1-MTG8 in leukemogenesis, we used oligonucleotide microarrays to detect alterations in gene expression caused by ectopic expression of AML1-MTG8 in a murine myeloid progenitor cell line, L-G. Microarray analysis of approximately 6500 genes identified 32 candidate genes under the downstream control of AML1-MTG8. Among the 32 genes, 23 were not known to be regulated by AML1-MTG8. These included many granule protein genes and several cell surface antigen genes. Interestingly, AML1-MTG8 enhanced the expression of several genes that are usually induced during granulocytic differentiation, particularly those encoding azurophil granule proteins, including cathepsin G, myeloperoxidase and lysozyme. This indicates that AML1-MTG8 induces partial differentiation of myeloid progenitor cells into promyelocytes in the absence of the usual differentiation signals, while it inhibits terminal differentiation into mature granulocytes. Thus, AML1-MTG8 itself may play a crucial role in defining a unique cytologic type with abnormal maturation, characteristic of t(8;21) acute myelogenous leukemia.

Developmental pathways are networks of genes that act coordinately to establish the body plan. Disruptions of genes in one pathway can have effects in related pathways and may result in serious dysmorphogenesis or cancer. Environmental exposures can be associated with poor pregnancy outcomes, including dysmorphic offspring or children with a variety of diseases. An important goal of environmental science should be reduction of these poor outcomes. This will require an understanding of the genes affected by specific exposures and the consequence of alterations in these genes or their products, which in turn will require an understanding of the pathways critical in development. The ligand Sonic hedgehog, the receptors Patched and Smoothened, and the GLI family of transcription factors represent one such pathway. This pathway illustrates several operating principles important in the consideration of developmental consequences of environmental exposures to toxins.

1. Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophila ether-à-go-go-like K+ channel (elk). 2. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3. Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage-activated K+ channels with novel properties. 4. RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at -90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG-mediated currents. 5. RELK2 channels produced currents with a fast inactivation component and HERG-like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

Mouse erythroleukemia (MEL) cell erythroid differentiation induced by dimethyl sulfoxide or hexamethylene bisacetamide (HMBA) is accompanied by the production of hemoglobin, terminal cell division and decreases in lactate production and fructose 2,6-bisphosphate levels. A number of studies have suggested that decreases in the cellular level of protein phosphotyrosine content may play a role in MEL cell differentiation. In particular, it was shown that the expression of several protein tyrosine phosphatase genes accompany this process and that the transfection of one of these genes into MEL cells followed by its subsequent expression induced eythroid differentiation. However, none of the physiological substrates for these protein tyrosine phosphatases have been identified. It is shown here that MEL cell differentiation is accompanied by decreases in tyrosine phosphorylation of Vav and possibly of the erythropoietin receptor (EpoR). Immunoprecipitation of the EpoR and analysis of co-precipitated proteins, indicates that the EpoR associates with Vav, STAT5 and an unidentified 60 Kd protein, . HMBA-induced erythroid differentiation abrogates these associations. The phosphatase inhibitors, Na3VO4 and levamisole, inhibit HMBA-induced differentiation as well as the association of the EpoR with Vav, STAT5 and the 60 Kd protein. This is of interest since Na3VO4, at the concentrations used here, has been shown to be a potent inhibitor of the activity of protein tyrosine phosphatases. These results suggest that levamisole, at least indirectly, acts by a molecular mechanism similar to that of Na3VO4 and that the loss of the association of the EpoR with Vav, STAT5, and and/or the reduction in the level of tyrosine phosphorylation of these proteins may play a role in MEL cell differentiation.

Retinoic acid (RA) inhibition of breast cancer cell growth is associated with an accumulation of cells in G1 phase of the cell cycle. We have investigated the effects of RA on the expression and activity of cell cycle-regulatory proteins in MCF-7 human breast cancer cells. Flow cytometry analysis of MCF-7 cells treated with RA revealed a decrease in the percentage of cells in S phase by 48 h, which was maximal by 72 h. Phosphorylation of the retinoblastoma protein (pRb) was partially reduced in RA-treated cells accompanied by a decrease in the level of retinoblastoma protein. Expression of the cyclin D1 transcript was reduced by 48 h and cyclin-dependent kinase 2 (cdk2) mRNA levels declined within 8 h posttreatment followed by a decrease in cyclin D1 and cdk2 protein levels. Message and protein levels of cdk4 and cdc2 were not affected by RA. While cdk4 activity was similar in control and RA-treated cells, cdk2 activity began to decrease within 48 h of exposure to RA and was profoundly reduced after 72 h. This reduced activity was associated with decreased phosphorylation of cdk2. The decrease in cdk2 activity occurred in the absence of RA-mediated increases in the levels of the cdk inhibitors p21 and p27. However, assays of cdk2 from pooled lysates from RA-treated and control cells showed that RA-treated cells contain a cdk2-inhibitory activity. Our results show that RA inhibits cell cycle progression of MCF-7 cells by inhibiting cdk2 mRNA and protein production and by decreasing cdk2 activity.

T cell activation stimulates p56(lck), p59(fyn), ZAP-70, Vav-SLP-76 binding, and IL-2 transcription. Major questions concern the tyrosine-kinase and relevant site(s) needed for Vav-SLP-76 complex formation and its role in IL-2 production. Here, we show that of the three kinases, only ZAP-70 phosphorylates SLP-76 at specific sites that allow Vav SH2 domain binding. Therefore, while p56(lck) regulates proximal events, ZAP-70 acts downstream on targets such as SLP-76. We also show by in vitro and in vivo analysis that two SLP-76 pYESP motifs (Y113 and Y128) mediate binding, the first being more efficient. A third pYEPP motif (Y145) failed to bind. Finally, TCR zeta CD3 ligation of T cell hybridoma DC27.10 induces IL-2 production without detectable Vav-SLP-76 binding. Therefore, despite effects of Vav-SLP-76 on IL-2 expression, Vav-SLP-76 binding per se is not essential for IL-2 production in all T cells.

SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck is a ubiquitously expressed adaptor protein whose function has been enigmatic. We performed confocal microscopy to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck was identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. Western blot analysis of nuclear and cytosolic fractions confirmed that there was no translocation in response to growth factor and that tyrosine phosphorylation was specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains revealed differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm.

Background: Alterations in the expression of genes that control the cell cycle may be of critical importance in determining the sensitivity of cells and tumors to drugs (chemosensitivity) and radiation. Mutations and deletions of the p53 tumor suppressor gene in cell lines and tumors are associated with resistance to a variety of DNA-damaging agents. The effects of alterations in the cyclin genes and their products on drug action have not been studied. One of these genes, cyclin D1, is expressed in early G1 phase, and its protein product, together with the cyclin-dependent kinases CDK4 and CDK6, mediates the phosphorylation and functional inactivation of the retinoblastoma protein (pRb). Elevated levels of expression of cyclin D1 protein have been found in a variety of cancers, including breast cancer, head and neck cancer, non-small-cell lung cancer, and mantle cell lymphomas., Purpose: This study was conducted to investigate the effect of increased expression of cyclin D1 protein on the chemosensitivity profile of a human fibrosarcoma cell line., Methods: Expression plasmids containing either the neomycin-resistance gene and the complementary DNA sequence encoding human cyclin D1 or the neomycin-resistance gene only (control) were transfected by lipofection into the human HT1080 fibrosarcoma cell line, and cell colonies resistant to the antibiotic neomycin (G418) were isolated. Cyclin D1 messenger RNA (mRNA) and protein levels were measured by ribonuclease protection and western blot analyses, respectively. Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively. The phosphorylation status of pRb was assessed by western blot analysis. Cell cycle analysis was performed by use of the technique of fluorescence-activated cell sorting. Cytotoxicity assays were carried out by use of the sulforhodamine blue assay., Results: Of the 16 cyclin D1-transfected cell clones that were isolated, four were randomly selected for further study. Two cell clones expressed high levels of cyclin D1 mRNA and protein as compared with control cells transfected with plasmids containing the neomycin-resistance gene only. A relative increase in the phosphorylated form of pRb in cells expressing high versus low levels of cyclin D1 was also revealed by western blot analysis. There was an increased fraction of cells in the S and G2 phases of the cell cycle among cells expressing higher levels of cyclin D1. Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression. Cytotoxicity assays revealed a statistically significant (P < .01) increase in resistance to methotrexate in cells expressing high levels of cyclin D1 compared with cells expressing lower levels. There was no difference in resistance to doxorubicin, paclitaxel (Taxol), and cytarabine., Conclusion: Alterations in the expression of cyclin D1 led to altered cell cycle distribution in a human sarcoma cell line. The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents., Implications: These results indicate that alterations in cell cycle genes may differ in their effects on cytotoxicity. It will be important to determine the effects of alterations of other cell cycle regulatory genes on the responses of cells to specific classes of drugs. Tumors with overexpression of cyclin D1 may be relatively refractory to methotrexate treatment.

Hepatocyte growth factor (HGF), a mesenchyme derived growth factor, promotes cell growth, cell motility, and morphogenesis in a variety of epithelial cells. The diverse responses are transduced across the cell membrane by the met/HGF receptor, a product of c-met protooncogene. The met/HGF receptor recruits a variety of second messenger molecules which relay the diverse intracellular responses of HGF. In this study, we show that HGF autophosphorylates and activates met/HGF receptor. The activated met/HGF receptor then physically associates with and activates phospholipase C-gamma (PLC-gamma). Furthermore, upon ligand stimulation, tyrosine-autophosphorylated met/HGF receptor also activates Nck oncogene product. Taken together, our results suggest that the receptor activation leads to formation of a complex in which PLC-gamma and Nck oncogene product co-exist with the activated met/HGF receptor, and that the Nck oncogene product is an important component of HGF signaling in Calu-1 and A549 cells.

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

Raji cells, a human Burkitt's lymphoma-derived cell line, will accumulate in a G0-like state upon prolonged (5-6 days) incubation in medium containing 1.5% dimethyl sulfoxide (DMSO). After removal of DMSO, the cells reenter the cell cycle in a synchronous manner and proliferate. After 5.5 days incubation in DMSO, S phase entry occurs at about 21-24 h after release, which is about the length of the first G1 phase of normal human lymphocytes which are stimulated in vitro to enter the cell cycle. The G0-like state of arrested cells and the sequence of events occurring after release from DMSO mimic, in most ways studied, those of normal lymphocytes. Arrested Raji cells lack many cell cycle-regulated molecules, including cyclin A, proliferating cell nuclear antigen, and the p34cdc2 kinase. They contain only hypophosphorylated p110Rb and a low level of enzymatically inactive p33cdk2 kinase. After reentering the cell cycle, a series of events occurred, including phosphorylation of p110Rb and accumulation of the cyclin A and proliferating cell nuclear antigen proteins in mid-G1 and the accumulation of the p33cdk2 and p34cdc2 proteins beginning in late G1, just prior to S-phase entry. Cyclin E levels in Raji cells appeared to be less regulated than in normal cells, with high levels of this protein being present in resting cells and throughout the entire cell cycle. The time courses of activation of the p34cdc2 and p33cdk2 kinases were similar; both became detectable at about 21 h after release and increased greatly in early S.(ABSTRACT TRUNCATED AT 250 WORDS)

Over the past 10 years, more than 40 potentially oncogenic genes, termed protooncogenes, have been identified in the human genome. Little is known of the physiological role of the proteins encoded by these genes, but they seem to be involved in the reception and transmission of hormonal and other environmental information from the cell membrane to the nucleus. These proteins may acquire transforming properties when over-expressed or if structurally altered following partial deletions or point mutations. Cytogenetic analysis shows loss of genetic material from specific chromosomal loci in many human tumors, suggesting that the absence of a functional gene at these loci may permit tumor development. The genes involved have been termed "anti-oncogenes". Understanding the control mechanisms of cell proliferation is essential in order to understand how cancer cells escape from this control. To this end, numerous oncogenes have been cloned, permitting the production of modified forms of oncogenic proteins and identification of the regions essential for their biological activity. Availability of large amounts of protein also allows the production of specific antibody which can be used to verify whether blockage of a given protein results in reversion of the transformed phenotype. If it can be shown that the expression of an oncogenic protein is essential for transformation, it should be possible to search for molecules that inhibit its action or which mimic the effects of an anti-oncogene. This type of research is already well advanced for the oncogenic ras proteins, and models have been established that permit both screening for potential inhibitors and design of specific antagonists.