NETWORK TOPOLOGY IN HUMAN PROTEIN INTERACTION DATA PREDICTS FUNCTIONAL ASSOCIATION

High-throughput assays, such as yeast two-hybrid system, have generated a huge amount of protein-protein interaction (PPI) data in the past decade. This tremendously increases the need for developing reliable methods to systematically and automatically suggest protein functions and relationships between them. With the available PPI data, it is now possible to study the functions and relationships in the context of a large-scale network. To data, several network-based schemes have been provided to effectively annotate protein functions on a large scale. However, due to those inherent noises in high-throughput data generation, new methods and algorithms should be developed to increase the reliability of functional annotations. Previous work in a yeast PPI network (Samanta and Liang, 2003) has shown that the local connection topology, particularly for two proteins sharing an unusually large number of neighbors, can predict functional associations between proteins, and hence suggest their functions. One advantage of the work is that their algorithm is not sensitive to noises (false positives) in high-throughput PPI data. In this study, we improved their prediction scheme by developing a new algorithm and new methods which we applied on a human PPI network to make a genome-wide functional inference. We used the new algorithm to measure and reduce the influence of hub proteins on detecting functionally associated proteins. We used the annotations of the Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) as independent and unbiased benchmarks to evaluate our algorithms and methods within the human PPI network. We showed that, compared with the previous work from Samanta and Liang, our algorithm and methods developed in this study improved the overall quality of functional inferences for human proteins. By applying the algorithms to the human PPI network, we obtained 4,233 significant functional associations among 1,754 proteins. Further comparisons of their KEGG and GO annotations allowed us to assign 466 KEGG pathway annotations to 274 proteins and 123 GO annotations to 114 proteins with estimated false discovery rates of <21% for KEGG and <30% for GO. We clustered 1,729 proteins by their functional associations and made pathway analysis to identify several subclusters that are highly enriched in certain signaling pathways. Particularly, we performed a detailed analysis on a subcluster enriched in the transforming growth factor β signaling pathway (P<10-50) which is important in cell proliferation and tumorigenesis. Analysis of another four subclusters also suggested potential new players in six signaling pathways worthy of further experimental investigations. Our study gives clear insight into the common neighbor-based prediction scheme and provides a reliable method for large-scale functional annotations in this post-genomic era.

CELL POLARITY REGULATES ORGAN GROWTH THROUGH THE HIPPO PATHWAY

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are linked to human cancer. Loss of polarity is highly correlated with malignancy. In Drosophila, perturbation of apical-basal polarity, including overexpressing the apical determinant Crumbs, can lead to uncontrolled tissue growth. Cells mutant for the basolateral determinant scribble overproliferate and can form neoplastic tumors. Interestingly, scribble mutant clones that arise in wild-type tissues are eliminated and therefore do not manifest their tumorigenic potential. However, the mechanisms by which cell polarity coordinates with growth control pathways in developing organs to achieve appropriate organ size remain obscure. To investigate the function of apical determinants in growth regulation, I investigated the mechanism by which the apical determinant Crumbs affects growth in Drosophila imaginal discs. I found that crumbs gain and loss of function cause overgrowth and induction of Hippo target genes. In addition, Crumbs is required for the proper localization of Expanded, an upstream component of the Hippo pathway. Furthermore, we uncoupled the cell polarity and growth control function of Crb through structure-functional analysis. Taken together, our data identify a role of Crb in growth regulation specifically through modulation of the Hippo pathway. To further explore the role of polarity in growth control, I investigated how cells mutant for basolateral determinants are eliminated by using patches of cells mutant for scribble (scribble mutant clones) as a model system. We found that competitive cell-cell interactions eliminate tumorigenic scribble cells by modulation of the Hippo pathway. The regulation of Hippo signaling is required and sufficient to restrain the tumorous growth of scribble mutant cells. Artificially increasing the relative fitness of scribble mutant cells unleashes their tumorigenic potential. Therefore, we have identified a novel tumor-suppression mechanism that depends on signaling between normal and tumorigenic cells. These data identify evasion of cell competition as a critical step toward malignancy and illustrate a role for wild-type tissue in eliminating abnormal cells and preventing the formation of tumors.

ROLE OF VITAMIN-D3 AND RETINOIC ACID IN A HUMAN THP-1 MACROPHAGE MODEL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D3 (VD) on macrophages in relation to the viability of Mtb. A combination of these vitamins (RAVD) enhanced the receptors on THP-1 macrophage (Mannose receptor and DC-SIGN) that increased mycobacterial uptake but inhibited thesubsequent intracellular growth of Mtb by inducing reactive oxygen species (ROS) and autophagy. RAVD also enhanced antigen presenting and homing receptors in THPs that suggested an activated phenotype for THPs following RAVD treatment. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent cells to formmultinucleate giant cells (MNGCs). Typically MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for as long as 60 days in culture. We propose that the RAVD mediated inhibition of replicating Mtb leading to persistence of non-replicating Mtb within THPs may provide a novel human macrophage model simulating formation of MNGCs in humanlungs.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis.

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia.

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT(1) receptor agonistic autoantibodies (AT(1)-AAs) that contribute to the disease features. However, the exact role of AT(1)-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT(1) receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT(1)-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT(1)-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT(1)-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT(1)-AA-induced placental damage. Our findings highlight AT(1)-AAs as important therapeutic targets.

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

THE MECHANISM OF TUMORIGENESIS IN THE IMMORTALIZED HUMAN PANCREATIC CELL LINES: CELL CULTURE MODELS OF HUMAN PANCREATIC CANCER

The mechanism of tumorigenesis in the immortalized human pancreatic cell lines: cell culture models of human pancreatic cancer Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer in the world. The most common genetic lesions identified in PDAC include activation of K-ras (90%) and Her2 (70%), loss of p16 (95%) and p14 (40%), inactivation p53 (50-75%) and Smad4 (55%). However, the role of these signature gene alterations in PDAC is still not well understood, especially, how these genetic lesions individually or in combination contribute mechanistically to human pancreatic oncogenesis is still elusive. Moreover, a cell culture transformation model with sequential accumulation of signature genetic alterations in human pancreatic ductal cells that resembles the multiple-step human pancreatic carcinogenesis is still not established. In the present study, through the stepwise introduction of the signature genetic alterations in PDAC into the HPV16-E6E7 immortalized human pancreatic duct epithelial (HPDE) cell line and the hTERT immortalized human pancreatic ductal HPNE cell line, we developed the novel experimental cell culture transformation models with the most frequent gene alterations in PDAC and further dissected the molecular mechanism of transformation. We demonstrated that the combination of activation of K-ras and Her2, inactivation of p16/p14 and Smad4, or K-ras mutation plus p16 inactivation, was sufficient for the tumorigenic transformation of HPDE or HPNE cells respectively. We found that these transformed cells exhibited enhanced cell proliferation, anchorage-independent growth in soft agar, and grew tumors with PDAC histopathological features in orthotopic mouse model. Molecular analysis showed that the activation of K-ras and Her2 downstream effector pathways –MAPK, RalA, FAK, together with upregulation of cyclins and c-myc were involved in the malignant transformation. We discovered that MDM2, BMP7 and Bmi-1 were overexpressed in the tumorigenic HPDE cells, and that Smad4 played important roles in regulation of BMP7 and Bmi-1 gene expression and the tumorigenic transformation of HPDE cells. IPA signaling pathway analysis of microarray data revealed that abnormal signaling pathways are involved in transformation. This study is the first complete transformation model of human pancreatic ductal cells with the most common gene alterations in PDAC. Altogether, these novel transformation models more closely recapitulate the human pancreatic carcinogenesis from the cell origin, gene lesion, and activation of specific signaling pathway and histopathological features.

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

Human placental lactogen enhancer: Tissue specificity and binding with specific proteins

Human placental lactogen (hPL) is a 22,000 dalton protein hormone produced in the placenta. The physiological actions of hPL are not well understood but its major activity is to regulate both maternal and fetal metabolism. hPL stimulates maternal lipolysis increasing free fatty acids in the maternal blood, allowing their use as an energy source by the mother, and sparing glucose for the fetus. It may also act as a growth promoting hormone for the fetus. hPL is produced in increasing amounts as pregnancy progresses. At term, hPL accounts for 10% of protein and 5% of total RNA in the placenta. This high level of hPL production is tissue-specific, as hPL is only produced in the placenta by syncytiotrophoblast cells.^ The objective of this work was to understand the mechanism by which such high levels of hPL are produced in a tissue-specific manner. A transcriptional enhancer found 2.2 kb 3$\sp\prime$ to one of the hPL genes (hPL$\sb3$) may explain the regulation of hPL expression. Transient transfection experiments using the hPL-producing human choriocarcinoma cell line JEG-3 localized the hPL enhancer to a 138 bp core element. This 138 bp sequence was found to be tissue specific in its actions as it did not promote transcription in heterologous cell lines. Gel mobility shift assays showed the hPL enhancer interacts specifically with nuclear proteins unique to hPL-producing cells. Within the 138 bp enhancer a 22 bp region was shown to be protected from DNase I digestion due to binding of proteins derived from placental nuclear extracts. Proteins binding this region of the enhancer may be instrumental in the tissue specific activity of the hPL enhancer. ^

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

Differential gene expression in multistep tumorigenesis using an {\it in vitro\/} human cell model

Using a human terato-carcinoma cell line, PA-1, the functional role of the oncogenes and tumor suppressor gene involved in the multistep process of carcinogenesis have been analyzed. The expression of AP-2 was strongly correlated with the susceptibility to ras transformation. The differential responsiveness to growth factors between stage 1 ras resistant cells and stage 2 ras susceptible cells was observed, indicating that the ability of stage 2 cells to respond to the mutated ras oncogenes in transformation correlated with the ability to be stimulated by certain growth factors. Using differential screening of cDNA libraries, a number of differentially expressed cDNA clones was isolated. One of those, clone 12, is overexpressed in ras transformed stage 3 cells. The amino acid sequence of clone 12 is almost identical to a mouse LLrep3 gene that was growth-regulated, and 78% similar to a yeast ribosomal protein S4. These results suggest that the S4 gene may be involved in regulation of growth. Clone 9 is expressed in stage 1 ras resistant cells (3.5-kb and 3.0-kb transcripts) but the expression of this clone in stage 2 ras susceptible cells and stage 3 ras-transformed cells is greatly diminished. The expression of this cDNA clone was increased to at least five fold in ras resistant cells and nontumorigenic hybrids treated with retinoic acid but not increased in retinoic acid treated ras susceptible cells, ras transformed cells and the tumorigenic segregants. Partial sequence of this clone showed no homology to the sequences in Genbank. These findings suggest that clone 9 could be a suppressor gene or the genes that are involved in the biochemical pathway of tumor suppression or neurogenic differentiation. The apparent pleiotropic effect of the loss of this suppressor gene function support Harris' proposal that tumor suppressor genes regulate differentiation. The tumor suppressor gene may act as negative regulator of tumor growth by controlling gene expression in differentiation. ^

Antisense-mediated inhibition of papillomavirus type-18 in human squamous cell carcinoma

Numerous co-factors, genetic, environmental and physical, play an important role in development and prognosis of cancer. Each year in the USA, more than 31,000 cases of oral and 13,000 cases of cervical cancer are diagnosed. Substantial epidemiological data supports a high correlation between development of these cancers and the presence of specific types of human papillomaviruses (HPV). Molecular biological studies show that not only are several of the viral genes necessary and sufficient to cause transformation but they also function synergistically with other co-factors. Evidence suggests that prevention of infection or inhibition of viral gene expression may alter the course of malignant transition. The main objective of this project was to test the hypothesis that some human carcinoma cells, containing HPV, behave in malignant manner because the viral genes function in the maintenance of some aspect of the transformed phenotype.^ The specific aims were (1) to select oral and cervical cancer cell lines which were HPV-negative or which harbored transcriptionally active HPV-18, (2) to construct and determine the effects of recombinant sense or antisense expressing vectors, (3) to test the effects of synthetic antisense oligodeoxynucleotides on the transformed behavior of these cells.^ To screen cells, we performed Southern and Northern analysis and polymerase chain reactions. When antisense-expressing vectors were used, cells harboring low numbers of HPV-18 where unable to survive transfection but they were readily transfected with all other constructs. Rare antisense transfectants obtained from HPV-positive cells showed significantly altered characteristics including malignant potential in nude mice. The HPV-negative cells showed no differences in transfection efficiencies or growth characteristics with any construct.^ In addition, treatment of the HPV-positive cells with antisense, but not random oligodeoxynucleotides, resulted in decreased cell proliferation and even cell death. These effects were dose-dependent, synergistic and HPV-specific.^ These results suggest that expression of viral genes play an important role in the maintenance of the transformed phenotype which implies that inhibition of expression, by antisense molecules, may be therapeutic in HPV-induced tumors. ^