Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^

TYPE I INSULIN-LIKE GROWTH FACTOR RECEPTOR TYROSINE KINASE AS A MOLECULAR TARGET IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoid malignancy representing 5-10% of all non-Hodgkin’s lymphomas. It is distinguished by the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the proto-oncogene CCND1, which encodes cyclin D1 at 11q13 to the IgH gene at 14q32. MCL patients represent about 6% of all new cases of Non-Hodgkin’s lymphomas per year or about 3,500 new cases per year. MCL occurs more frequently in older adults – the average age at diagnosis is the mid-60s with a male-to-female ratio of 2-3:1. It is typically characterized by the proliferation of neoplastic B-lymphocytes in the mantle zone of the lymph node follicle that have a prominent inclination to disseminate to other lymphoid tissues, bone marrow, peripheral blood and other organs. MCL patients have a poor prognosis because they develop resistance/relapse to current non-specific therapeutic regimens. It is of note that the exact molecular mechanisms underlying the pathogenesis of MCL are not completely known. It is reasonable to anticipate that better characterization of these mechanisms could lead to the development of specific and likely more effective therapeutics to treat this aggressive disease. The type I insulin-like growth factor receptor (IGF-IR) is thought to be a key player in several different solid malignancies such as those of the prostate, breast, lung, ovary, skin and soft tissue. In addition, recent studies in our lab showed evidence to support a pathogenic role of IGF-IR in some types of T-cell lymphomas and chronic myeloid leukemia. Constitutively active IGF-IR induces its oncogenic effects through the inhibition of apoptosis and induction of transformation, metastasis, and angiogenesis. Previous studies have shown that signaling through IGF-IR leads to the vi activation of multiple signaling transduction pathways mediated by the receptor-associated tyrosine kinase domain. These pathways include PI3K/Akt, MAP kinase, and Jak/Stat. In the present study, we tested the possible role of IGF-IR in MCL. Our results demonstrate that IGF-IR is over-expressed in mantle cell lymphoma cell lines compared with normal peripheral blood B- lymphocytes. Furthermore, inhibition of IGF-IR by the cyclolignan picropodophyllin (PPP) decreased cell viability and cell proliferation in addition to induction of apoptosis and G2/M cell cycle arrest. Screening of downstream oncogenes and apoptotic proteins that are involved in both IGF-IR and MCL signaling after treatment with PPP or IGF-IR siRNA showed significant alterations that are consistent with the cellular changes observed after PPP treatment. Therefore, our findings suggest that IGF-IR signaling contributes to the survival of MCL and thus may prove to be a legitimate therapeutic target in the future.

Molecular mechanisms of prostatic carcinoma growth and progression

Prostate cancer represents the most commonly diagnosed malignancies in American men and is the second leading cause of male cancer deaths. The overall objectives of this research were designed to understand the cellular and molecular mechanisms of prostatic carcinoma growth and progression. This dissertation was divided into two major parts: (1) to clone and characterize soluble factor(s) associated with bone that may mediate prostatic carcinoma growth and progression; (2) to investigate the roles of extracellular matrix in prostatic carcinogenesis.^ The propensity of prostate cancer cells to metastasize to the axial skeleton and the subsequent osteoblastic reactions observed in the bone indicate the possible reciprocal cellular interaction between prostate cancer cells and the bone microenvironment. To understand the molecular and cellular basis of this interaction, I focused on the identification and cloning of soluble factor(s) from bone stromal cells that may exert direct mitogenic action on cultured prostate cells. A novel BPGF-1 gene expressed specifically by bone and male accessory sex organs (prostate, seminal vesicles, and coagulating gland) was identified and cloned.^ The BPGF-1 was identified and cloned from a cDNA expression library prepared from a human bone stromal cell line, MS. The conditioned medium (CM) of this cell line contains mitogenic materials that induce human prostate cancer cell growth both in vivo and in vitro. The cDNA expression library was screened by an antibody prepared against the mitogenic fraction of the CM.^ The cloned BPGF-1 cDNA comprises 3171 nucleotides with a single open reading frame of 1620 nucleotides encoding 540 amino acids. The BPGF-1 gene encodes two transcripts (3.3 and 2.5 kb) with approximately equal intensity in human cells and tissues, but only one transcript (2.5 kb) in rat and mouse tissues. Southern blot analysis of human genomic DNA revealed a single BPGF-1 gene. The BPGF-1 gene is expressed predominantly in bone and seminal vesicles, but at a substantially lower level in prostate. Polyclonal antibodies generated from synthetic peptides that correspond to the nucleotide sequences of the cloned BPGF-1 cDNA reacted with a putative BPGF-1 protein with an apparent molecular weight of 70 kDa. The conditioned media isolated from COS cells transfected with BPGF-1 cDNA stimulated the proliferation and increased the anchorage-independent growth of prostate epithelial cells. These findings led us to hypothesize that BPGF-1 expression in relevant organs, such as prostate, seminal vesicles, and bone, may lead to local prostate cancer growth, metastasis to the seminal vesicles, and subsequently dissemination to the skeleton.^ To assess the importance of extracellular matrix in prostatic carcinogenesis, the role of extracellular matrix in induction of rat prostatic carcinoma growth in vivo was evaluated. NbE-1, a nontumorigenic rat prostatic epithelial cell line, was induced to form carcinoma in athymic nude hosts by coinjecting them with Matrigel and selected extracellular matrix components. Induction of prostatic tumor formation by laminin and collagen IV was inhibited by their respective antibodies. Prostatic epithelial cells cloned from the tumor tissues were found to form tumors in athymic nude hosts in the absence of exogenously added extracellular matrix. These results suggest that extracellular matrix induce irreversibly prostatic epithelial cells that behave distinctively different from the parental prostatic epithelial cell line. ^

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Computer simulation of glioma growth and morphology.

Despite major advances in the study of glioma, the quantitative links between intra-tumor molecular/cellular properties, clinically observable properties such as morphology, and critical tumor behaviors such as growth and invasiveness remain unclear, hampering more effective coupling of tumor physical characteristics with implications for prognosis and therapy. Although molecular biology, histopathology, and radiological imaging are employed in this endeavor, studies are severely challenged by the multitude of different physical scales involved in tumor growth, i.e., from molecular nanoscale to cell microscale and finally to tissue centimeter scale. Consequently, it is often difficult to determine the underlying dynamics across dimensions. New techniques are needed to tackle these issues. Here, we address this multi-scalar problem by employing a novel predictive three-dimensional mathematical and computational model based on first-principle equations (conservation laws of physics) that describe mathematically the diffusion of cell substrates and other processes determining tumor mass growth and invasion. The model uses conserved variables to represent known determinants of glioma behavior, e.g., cell density and oxygen concentration, as well as biological functional relationships and parameters linking phenomena at different scales whose specific forms and values are hypothesized and calculated based on in vitro and in vivo experiments and from histopathology of tissue specimens from human gliomas. This model enables correlation of glioma morphology to tumor growth by quantifying interdependence of tumor mass on the microenvironment (e.g., hypoxia, tissue disruption) and on the cellular phenotypes (e.g., mitosis and apoptosis rates, cell adhesion strength). Once functional relationships between variables and associated parameter values have been informed, e.g., from histopathology or intra-operative analysis, this model can be used for disease diagnosis/prognosis, hypothesis testing, and to guide surgery and therapy. In particular, this tool identifies and quantifies the effects of vascularization and other cell-scale glioma morphological characteristics as predictors of tumor-scale growth and invasion.

Predictive oncology: a review of multidisciplinary, multiscale in silico modeling linking phenotype, morphology and growth.

Empirical evidence and theoretical studies suggest that the phenotype, i.e., cellular- and molecular-scale dynamics, including proliferation rate and adhesiveness due to microenvironmental factors and gene expression that govern tumor growth and invasiveness, also determine gross tumor-scale morphology. It has been difficult to quantify the relative effect of these links on disease progression and prognosis using conventional clinical and experimental methods and observables. As a result, successful individualized treatment of highly malignant and invasive cancers, such as glioblastoma, via surgical resection and chemotherapy cannot be offered and outcomes are generally poor. What is needed is a deterministic, quantifiable method to enable understanding of the connections between phenotype and tumor morphology. Here, we critically assess advantages and disadvantages of recent computational modeling efforts (e.g., continuum, discrete, and cellular automata models) that have pursued this understanding. Based on this assessment, we review a multiscale, i.e., from the molecular to the gross tumor scale, mathematical and computational "first-principle" approach based on mass conservation and other physical laws, such as employed in reaction-diffusion systems. Model variables describe known characteristics of tumor behavior, and parameters and functional relationships across scales are informed from in vitro, in vivo and ex vivo biology. We review the feasibility of this methodology that, once coupled to tumor imaging and tumor biopsy or cell culture data, should enable prediction of tumor growth and therapy outcome through quantification of the relation between the underlying dynamics and morphological characteristics. In particular, morphologic stability analysis of this mathematical model reveals that tumor cell patterning at the tumor-host interface is regulated by cell proliferation, adhesion and other phenotypic characteristics: histopathology information of tumor boundary can be inputted to the mathematical model and used as a phenotype-diagnostic tool to predict collective and individual tumor cell invasion of surrounding tissue. This approach further provides a means to deterministically test effects of novel and hypothetical therapy strategies on tumor behavior.

THE ROLE AND MECHANISM OF THE HOMEOBOX GENE DLX4 IN TRANSFORMING GROWTH FACTOR-B RESISTANCE IN CANCER

Transforming growth factor-b (TGF-b) is a cytokine that plays essential roles in regulating embryonic development and tissue homeostasis. In normal cells, TGF-b exerts an anti-proliferative effect. TGF-b inhibits cell growth by controlling a cytostatic program that includes activation of the cyclin-dependent kinase inhibitors p15Ink4B and p21WAF1/Cip1 and repression of c-myc. In contrast to normal cells, many tumors are resistant to the anti-proliferative effect of TGF-b. In several types of tumors, particularly those of gastrointestinal origin, resistance to the anti-proliferative effect of TGF-b has been attributed to TGF-b receptor or Smad mutations. However, these mutations are absent from many other types of tumors that are resistant to TGF-b-mediated growth inhibition. The transcription factor encoded by the homeobox patterning gene DLX4 is overexpressed in a wide range of malignancies. In this study, I demonstrated that DLX4 blocks the anti-proliferative effect of TGF-b by disabling key transcriptional control mechanisms of the TGF-b cytostatic program. Specifically, DLX4 blocked the ability of TGF-b to induce expression of p15Ink4B and p21WAF1/Cip1 by directly binding to Smad4 and to Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc, a repressor of p15Ink4B and p21WAF1/Cip1 transcription, independently of TGF-b signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-b cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-b. This study provides a molecular explanation as to why tumors are resistant to the anti-proliferative effect of TGF-b in the absence of mutations in the TGF-b signaling pathway. Furthermore, this study also provides insights into how aberrant activation of a developmental patterning gene promotes tumor pathogenesis.

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.

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

DOWNREGULATION OF PAX2 SUPPRESSES OVARIAN CANCER CELL GROWTH

PAX2 is one of nine PAX genes regulating tissue development and cellular differentiation in embryos. PAX2 promotes cell proliferation, oncogenic transformation, cell-lineage specification, migration, and survival. Unattenuated PAX2 has been found in several cancer types. We therefore sought to elucidate the role of PAX2 in ovarian carcinomas. We found that PAX2 was expressed in low-grade serous, clear cell, endometrioid and mucinous cell ovarian carcinomas, which are relatively chemoresistant compared to high grade serous ovarian carcinomas. Four ovarian cancer cell lines, RMUGL (mucinous), TOV21G (clear cell), MDAH-2774 (endometrioid) and IGROV1 (endometrioid), which express high-levels of PAX2, were used to study the function of PAX2. Lentiviral shRNAs targeting PAX2 were used to knock down PAX2 expression in these cell lines. Cellular proliferation and motility assays subsequently showed that PAX2 stable knockdown had slower growth and migration rates. Microarray gene expression profile analysis further identified genes that were affected by PAX2 including the tumor suppressor gene G0S2. Reverse phase protein array (RPPA) data showed that PAX2 knockdown affected several genes that are involved in apoptosis, which supports the fact that downregulation of PAX2 in PAX2-expressing ovarian cancer cells inhibits cell growth. We hypothesize that this growth inhibition is due to upregulation of the tumor suppressor gene G0S2 via induction of apoptosis. PAX2 represents a potential therapeutic target for chemoresistant PAX2-expressing ovarian carcinomas.

Contribution of Ectodomain Mutations in Epidermal Growth Factor Receptor to Signaling in Glioblastoma Multiforme

CONTRIBUTION OF ECTODOMAIN MUTATIONS IN EPIDERMAL GROWTH FACTOR RECEPTOR TO SIGNALING IN GLIOBLASTOMA MULTIFORME Publication No._________ Marta Rojas, M.S. Supervisory Professor: Oliver Bögler, Ph.D. The Cancer Genome Atlas (TCGA) has conducted a comprehensive analysis of a large tumor cohort and has cataloged genetic alterations involving primary sequence variations and copy number aberrations of genes involved in key signaling pathways in glioblastoma (GBM). This dataset revealed missense ectodomain point mutations in epidermal growth factor receptor (EGFR), but the biological and clinical significance of these mutations is not well defined in the context of gliomas. In our study, we focused on understanding and defining the molecular mechanisms underlying the functions of EGFR ectodomain mutants. Using proteomic approaches to broadly analyze cell signaling, including antibody array and mass spectrometry-based methods, we found a differential spectrum of tyrosine phosphorylation across the EGFR ectodomain mutations that enabled us to stratify them into three main groups that correlate with either wild type EGFR (EGFR) or the long-studied mutant, EGFRvIII. Interestingly, one mutant shared characteristics of both groups suggesting a continuum of behaviors along which different mutants fall. Surprisingly, no substantial differences were seen in activation of classical downstream signaling pathways such as Akt and S6 pathways between these classes of mutants. Importantly, we demonstrated that ectodomain mutations lead to differential tumor growth capabilities in both in vitro (anchorage independent colony formation) and in vivo conditions (xenografts). Our data from the biological characterization allowed us to categorize the mutants into three main groups: the first group typified by EGFRvIII are mutations with a more aggressive phenotype including R108K and A289T; a second group characterized by a less aggressive phenotype exemplified by EGFR and the T263P mutation; and a third group which shared characteristics from both groups and is exemplified by the mutation A289D. In addition, we treated cells overexpressing the mutants with various agents employed in the clinic including temozolomide, cisplatin and tarceva. We found that cells overexpressing the mutants in general displayed resistance to the treatments. Our findings yield insights that help with the molecular characterization of these mutants. In addition, our results from the drug studies might be valuable in explaining differential responses to specific treatments in GBM patients.

A molecular analysis of the conditional defect in MuSVts 110 viral RNA splicing

Cells infected with MuSVts110 express a viral RNA which contains an inherent conditional defect in RNA splicing. It has been shown previously that splicing of the MuSVts110 primary transcript is essential to morphological transformation of 6m2 cells in vitro. A growth temperature of 33$\sp\circ$C is permissive for viral RNA splicing,and, consequently, 6m2 cells appear morphologically transformed at this temperature. However, 6m2 cells appear phenotypically normal when incubated at 39$\sp\circ$C, the non-permissive temperature for viral RNA splicing.^ After a shift from 39$\sp\circ$C to 33$\sp\circ$C, the coordinate splicing of previously synthesized and newly transcribed MuSVts110 RNA was achieved. By S1 nuclease analysis of total RNA isolated at various times, 5$\sp\prime$ splice site cleavage of the MuSVts110 transcript appeared to occur 60 minutes after the shift to 33$\sp\circ$C, and 30 minutes prior to detectable exon ligation. In addition, consistent with the permissive temperatures and the kinetic timeframe of viral RNA splicing after a shift to 33$\sp\circ$C, four temperature sensitive blockades to primer extension were identified 26-75 bases upstream of the 3$\sp\prime$ splice site. These blockades likely reflect four branchpoint sequences utilized in the formation of MuSVts110 lariat splicing-intermediates.^ The 54-5A4 cell line is a spontaneous revertant of 6m2 cells and appears transformed at all growth temperatures. Primer extension sequence analysis has shown that a five base deletion occurred at the 3$\sp\prime$ splice site in MuSVts110 RNA allowing the expression of a viral transforming protein in 54-5A4 in the absence of RNA splicing, whereas in the parental 6m2 cell line, a splicing event is necessary to generate a similar transforming protein. As a consequence of this deletion, splicing cannot occur and the formation of the four MuSVts110 branched-intermediates were not observed at any temperature in 54-5A4 cells. However, 5$\sp\prime$ splice site cleavage was still detected at 33$\sp\circ$C.^ Finally, we have investigated the role of the 1488 bp deletion which occurred in the generation of MuSVts110 in the activation of temperature sensitive viral RNA splicing. This deletion appears solely responsible for splice site activation. Whether intron size is the crucial factor in MuSVts110 RNA splicing or whether inhibitory sequences were removed by the deletion is currently unknown. (Abstract shortened with permission of author.) ^

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Molecular signals in B lymphocyte activation: The role of intracellular calcium ion concentration, protein kinase C activation, and autocrine interleukin-2

Calcium ionophore, ionomycin, and phorbol myristate acetate (PMA) were used to activate rabbit peripheral blood B cells to study the role of increased intracellular calcium ion concentration ( (Ca$\sp2+\rbrack\sb{\rm i}$), protein kinase C (PKC) activation, and autocrine interleukin (IL-2) in inducing cell cycle entry and maintaining activation to DNA synthesis. When stimulated with a combination of ionomycin and PMA the B cells produced a soluble factor that supported the IL-2 dependent cell line, CTLL-2. The identity of the factor was established as IL-2 and its source was proved to be B cells in further experiments. Absorption studies and limiting dilution analysis indicated that IL-2 produced by B cells can act as an autocrine growth factor. Next, the effect of complete and incomplete signalling on B lymphocyte activation leading to cell cycle entry, IL-2 production, functional IL-2 receptor (IL-2R) expression, and DNA synthesis was examined. It was observed that cell cycle entry could be induced by signals provided by each reagent alone, but IL-2 production, IL-2R expression, and progression to DNA synthesis required activation with both reagents. Incomplete activation with ionomycin or PMA alone altered the responsiveness of B cells to further stimulation only in the case of ionomycin, and the unresponsiveness of these cells was apparently due to a lack of functional IL-2R expression on these cells, even though IL-2 production was maintained. The requirement of IL-2 for maintenance of activation to DNA synthesis was then investigated. The hypothesis that IL-2, acts in late G$\sb1$ and is required for DNA synthesis in B cells was supported by comparing IL-2 production and DNA synthesis in peripheral blood cells and purified B cells, kinetic analysis of these events in B cells, effects of anti-IL-2 antibody and PKC inhibitors, and by the response of G$\sb1$ B cells. Additional signals transduced by the interaction of autocrine IL-2 and functional IL-2 receptor on rabbit B cells were found to be necessary to drive these cells to S phase, after initial activation caused by simultaneous increase in (Ca$\sp2+\rbrack\sb{\rm i}$ and PKC activation had induced cell cycle entry, IL-2 production, and functional IL-2 receptor expression. ^