THE ROLE OF MAP KINASES ON THE FUNCTIONAL HETEROGENEITY OF HUMAN CD8+ T CELL MATURATION SUBSETS

While prior studies have focused on naïve (CD45RA+CD27+) and early stage memory (CD45RA-CD27+) CD8+ T cells, late memory CD8+ T cells (CD45RA+CD27) have received less interest because this subset of T cells is generally recognized as effectors, which produce IFNγ (but no IL-2) and perforin. However, multiple studies suggest that late memory CD8+ T cells may provide inadequate protection in infectious diseases and cancer models. To better understand the unique function of late memory CD8+ T cells, I optimized multi-color flow cytometry techniques to assess the cytokine production of each human CD8+ T cell maturation subset. I demonstrated that late memory CD8+ T cells are the predominant producer of CC chemokines (e.g. MIP-1β), but rarely produce IL-2; therefore they do not co-produce IL-2/IFNγ (polyfunctionality), which has been shown to be critical for protective immunity against chronic viral infection. These data suggest that late memory CD8+ T cells are not just cytotoxic effectors, but may have unique functional properties. Determining the molecular signature of each CD8+ T cell maturation subset will help characterize the role of late memory CD8+ T cells. Prior studies suggest that ERK1 and ERK2 play a role in cytokine production including IL-2 in T cells. Therefore, I tested whether differential expression of ERK1 and ERK2 in CD8+ T cell maturation subsets contributes to their functional signature by a novel flow cytometry technique. I found that the expression of total ERK1, but not ERK2, is significantly diminished in late memory CD8+ T cells and that ERK1 expression is strongly associated with IL-2 production and CD28 expression. I also found that IL-2 production is increased in late memory CD8+ T cells by over-expressing ERK1. Collectively, these data suggest that ERK1 is required for IL-2 production in human CD8+ T cells. In summary, this dissertation demonstrated that ERK1 is down-regulated in human late memory CD8+ T cells, leading to decreased production of IL-2. The data in this dissertation also suggested that the functional heterogeneity in human CD8+ T cell maturation subsets results from their differential ERK1 expression.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy.

TNF-α (tumor necrosis factor-α) is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL) in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1) mechanical properties, employing atomic force microscopy; 2) cytoskeletal organization, through fluorescence microscopy; and 3) membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h), for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells); the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with controls. Collectively, these results demonstrate that sufficiently high levels of circulating TNF-α have similar effects on different endothelial districts, and provide additional information for unraveling the possible correlations between circulating pro-inflammatory cytokines and systemic vascular dysfunction.

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. ^

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Federal regulation of human embryonic stem cell research

President George W. Bush's 2001 statement, which laid out guidelines for research that uses human embryonic stem cells to qualify for federal funding, intends to prevent new embryonic stem cell lines from being developed, by prohibiting the federal funding of research that uses embryonic stem cell lines other than those that existed at the time of the policy's inception and were approved by the National Institutes of Health. This policy raises questions of medical and technological ethics and the governments' role in making decisions regarding the advancement of science based on moral and political opinions. Federal stem cell usage policy directly affects scientific research efforts that are currently on the path to understanding the mechanisms of cell differentiation and could potentially offer answers and therapies for disabilities and many chronic diseases. By reviewing the current literature on the background information on human embryonic stem cells, including what they are, where they come from, how they are used for research purposes, and the ethical controversy surrounding their use, I have researched and reported the impact of the 2001 policy on medical research. ^ Both those who support the current policy on human embryonic stem cell research and those who are advocates for policy change have relevant arguments and varying opinions on human embryonic stem cell usage itself. The ethical implication of how embryonic stem cells are obtained has led to fierce debate. This paper presents many arguments for and against hESC research in addition to the policy governing their use. This analysis concludes that the current policy on federal funding of human embryonic stem cell research should be revised to allow research using new stem lines to be eligible for federal funding under specific guidelines. Supporting evidence for this recommendation is provided.^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Regulation of vascular endothelial growth factor expression in human pancreatic cancer

Pancreatic adenocarcinoma is currently the fifth-leading cause of cancer-related death in the United States. Like with other solid tumors, the growth and metastasis of pancreatic adenocarcinoma are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in angiogenesis, growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. However, the expression and regulation of VEGF in human pancreatic cancer cells are mostly unknown. ^ To examine the hypothesis that VEGF is constitutively expressed in human pancreatic cancer cells, and can be further induced by tumor environment factors such as nitric oxide, a panel of human pancreatic cancer cell lines were studied for constitutive and inducible VEGF expression. All the cell lines examined were shown to constitutively express various levels of VEGF. To identify the mechanisms responsible for the elevated expression of VEGF, its rates of turnover and transcription were then investigated. While the half-live of VEGF was unaffected, higher transcription rates and increased VEGF promoter activity were observed in tumor cells that constitutively expressed elevated levels of VEGF. Detailed VEGF promoter analyses revealed that the region from −267 to +50, which contains five putative Sp1 binding sites, was responsible for this VEGF promoter activity. Further deletion and point mutation analyses indicated that deletion of any of the four proximal Sp1 binding sites significantly diminished VEGF promoter activity and when all four binding sites were mutated, it was completely abrogated. Consistent with these observations, high levels of constitutive Sp1 expression and DNA binding activities were detected in pancreatic cancer cells expressing high levels of VEGF. Collectively, our data indicates that constitutively expressed Sp1 leads to the constitutive expression of VEGF, and implicates that both molecules involve in the aggressive pathogenesis of human pancreatic cancer. ^ Although constitutively expressed in pancreatic cancer cells, VEGF can be further induced. In human pancreatic cancer specimens, we found that in addition to VEGF, both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were overexpressed, suggesting that nitric oxide might upregulate VEGF expression. Indeed, a nitric oxide donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) significantly induced VEGF mRNA expression and protein secretion in pancreatic adenocarcinoma cells in a time- and dose-dependant manner. Using a luciferase reporter containing both the VEGF promoter and the 3′ -UTR, we showed that SNAP significantly increased luciferase activity in human pancreatic cancer cells. Notwithstanding its ability to induce VEGF in vitro, pancreatic cancer cells genetically engineered to produce NO did not exhibit increased tumor growth. This inability of NO to promote tumor growth appears to be related to NO-mediated cytotoxicity. The balance between NO mediated effects on pro-angiogenesis and cytotoxicity would determine the biological outcome of NO action on tumor cells. ^ In summary, we have demonstrated that VEGF is constitutively expressed in human pancreatic cancer cells, and that overexpression of transcription factor Sp1 is primarily responsible. Although constitutively expressed in these cells, VEGF can be further induced by NO. However, using a mouse model, we have shown that NO inhibited tumor growth by promoting cytotoxicity. These studies suggest that both Sp1 and NO may be important targets for designing potentially effective therapies of human pancreatic cancer and warrant further investigation. ^

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.

Host factors in metastasis: Direct lysis of bloodborne metastatic tumor cells by murine vascular endothelial cells

During the process of cancer metastasis, the majority of circulating tumor cells arrest in microcapillary beds and then rapidly die. To study whether vascular endothelial cells can directly lyse tumor cells, we isolated vascular endothelial cells by perfusion of lungs from immunocompetent or nude mice. The cells were grown in culture, and then cloned and characterized. Cloned endothelial cells were incubated with several lymphokines and cytokines. Cells incubated with IFN-$\gamma$ and TNF lysed a variety of tumor cells with different metastatic potential. Mouse skin and lung fibroblasts treated with the same cytokines did not. Endothelial cell mediated tumor cell lysis was not due to different binding ability of tumor cells to cytokine treated and untreated endothelial monolayers. Kinetic studies demonstrated that the continuous presence of cytokines in the tumor-endothelial cocultures was necessary to produce maximal lysis of tumor cells. Target cell lysis was not due to the direct effects of IFN-$\gamma$ or TNF, since vascular endothelial cells isolated from the lung of nude mice lysed human melanoma cells that are sensitive or resistant to TNF. Cytokine treated endothelial cells produced a high level of nitric oxide, which is known to be cytotoxic to a variety of target cells. The level of nitric oxide production was directly correlated with the degree of tumor cell lysis. A specific inhibitor of nitric oxide synthesis(N$\sp{\rm G}$-monomethyl-L-arginine), completely inhibited production of nitric oxide and tumor cell lysis. Treatment of cytokine activated endothelial cells with dexamethasone also inhibited tumor cell lysis. This inhibition was independent of tumor-endothelial adhesion but correlated with inhibition of nitric oxide production. Collectively, these results suggest that vascular endothelial cells can directly destory tumor emboli and thus play an active role in the pathogenesis of cancer metastasis. ^

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. ^