AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4388 CD147 is a widely expressed transmemb... more AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4388 CD147 is a widely expressed transmembrane glycoprotein that is upregulated on cancer cells. Over-expression of CD147, a member of the immunoglobulin super-family, promotes invasion, metastasis, and growth and survival of malignant cells. The molecular mechanisms underlying the actions of CD147 are not fully understood. We utilized fibroblast cells from wild-type and CD147 null mice to study signal transduction pathways activated downstream of CD147. When CD147 null fibroblasts were exposed to UV light we observed defective activation of JNK compared to control CD147 expressing fibroblasts. Since CD147 is a cell surface glycoprotein involved in cell-cell interactions and is thought to interact with ligands such as cyclophilins or to interact homotypically or potentially with unknown cell surface proteins on opposing cells, we ligated CD147 with mAb to examine activation of signaling proteins downstream of CD147. We observed that Akt, c-Jun Kinase (JNK), and Erk1/2 are all activated following treatment of CD147 expressing fibroblasts with anti-CD147 mAb OX114. We treated cells with inhibitors of PI3 Kinase (LY294002) and MEK (PD98059) to determine the importance of these signaling pathways in CD147 mediated activation. Inhibition of PI3 Kinase inhibited activation of Akt that follows CD147 ligation, indicating that activation of PI3 Kinase is required for CD147 induced Akt activation. Similarly, c-Jun Kinase activation was greatly decreased by the PI3 Kinase inhibitor. Although the MEK inhibitor did not completely inhibit Erk1/2 activation, it greatly decreased c-Jun Kinase activation indicating that MEK is required for c-Jun Kinase activation downstream of CD147 ligation. Increased expression of CD147 has been suggested to result in enhanced survival of transformed cells. Together with our results of increased Akt, Erk and JNK phosphorylation in response to CD147 ligation, a theme of CD147 promoting a survival signal is emerging. These CD147 signal transduction studies indicate that tumor cell survival may be linked not only to induction of matrix metalloproteinases, but to downstream signaling through CD147 as well.
Proceedings of the National Academy of Sciences of the United States of America, Feb 1, 1989
Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its... more Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its physiological function is unknown, but in vitro studies suggest that Thy-1 may transmit activation signals to T cells and may play a role in the growth and/or differentiation of thymocytes [
Proceedings of the National Academy of Sciences of the United States of America, Apr 14, 1998
Differential expression of surface markers can frequently be used to distinguish functional subse... more Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of V8.1 T cell antigen receptor (TCR) transgenic mice with a V8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1 a ) induces tolerance to Mls-1 a by clonal anergy. CD4 lymph node T cells from Mls-1 a inoculated transgenic mice enriched for the 6C10 ؊ phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10 ؉ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10 ؊ T cells remain 3G11 hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.
Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial... more Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.
This review is primarily concerned with unusual subsets of thymus-derived lymphocytes which our l... more This review is primarily concerned with unusual subsets of thymus-derived lymphocytes which our laboratory has analyzed. One of the many difficulties encountered in the study of T-cell differentiation pathways is that the actual numbers of T-lineage cells at a given stage of development is limited. Several methods have been conceived to overcome these difilculties, including establishment of precursor T-cell clones, thymocyte tumors or hybridomas, and microculture systems. Our laboratory has taken advantage of the gid mutation, which has dramatic effects on lymphocyte accumulation and positioning patterns. We will briefly discuss T-cell differentiation in general and then describe our experiments concerning novel T-cell subsets in peripheral lymph nodes of C3Hgld/gld mice. Finally, we will discuss our views concerning the origins of these novel T-ceil subsets and their relation to normal thymic differentiation.
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 1992
The bcI-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to ex... more The bcI-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend ceflular lifespan through Inhibition of apoptosis, or pro- grammed cell death. However, the physiological role of the Bcd-2 protein In lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcd-2 protein in both cortical and medulary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resist- ant to apoptosis mediated by corticosterolds and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcI-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.
Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxyge... more Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxygen species (ROS). We cloned the cDNA encoding the mouse homolog of TPx from an E14.5 brain cDNA library and analyzed its distribution and function in murine tissues. Comparison of the amino acid sequence of mouse TPx with those of other species revealed that TPx was highly conserved across all species. Mouse TPx had broad tissue distribution, but its expression was especially marked in cells that metabolize oxygen molecules at high levels such as erythroid cells, renal tubular cells, cardiac and skeletal muscle cells, and certain types of neurons. Levels of increased expression of TPx in the brain were coincident with regions known to be especially sensitive to hypoxic and ischemic injury in humans. Models of erythroid differentiation and neuronal survival were employed to study the function of TPx. Murine erythroleukemia cells (MEL cells) increased TPx transcription when in a chemically differentiated state. Furthermore, expression of mouse TPx in PC12 pheochromocytoma cells prolonged their survival in the absence of nerve growth factor (NGF) and serum, indicating that TPx could promote neuronal cell survival. We propose that TPx contributes to antioxidant defense in erythrocytes and neuronal cells by limiting the destructive capacity of oxygen radicals. These findings identify a novel gene that appears to be relevant to hypoxic brain injury and may be of importance in development of new approaches to abrogate the effects of ischemic- and hypoxic-related injury in the central nervous system (CNS).
p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our rece... more p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (T reg ) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185 her2/neu (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit T reg functions in targeted therapies for cancers.
AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4388 CD147 is a widely expressed transmemb... more AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA 4388 CD147 is a widely expressed transmembrane glycoprotein that is upregulated on cancer cells. Over-expression of CD147, a member of the immunoglobulin super-family, promotes invasion, metastasis, and growth and survival of malignant cells. The molecular mechanisms underlying the actions of CD147 are not fully understood. We utilized fibroblast cells from wild-type and CD147 null mice to study signal transduction pathways activated downstream of CD147. When CD147 null fibroblasts were exposed to UV light we observed defective activation of JNK compared to control CD147 expressing fibroblasts. Since CD147 is a cell surface glycoprotein involved in cell-cell interactions and is thought to interact with ligands such as cyclophilins or to interact homotypically or potentially with unknown cell surface proteins on opposing cells, we ligated CD147 with mAb to examine activation of signaling proteins downstream of CD147. We observed that Akt, c-Jun Kinase (JNK), and Erk1/2 are all activated following treatment of CD147 expressing fibroblasts with anti-CD147 mAb OX114. We treated cells with inhibitors of PI3 Kinase (LY294002) and MEK (PD98059) to determine the importance of these signaling pathways in CD147 mediated activation. Inhibition of PI3 Kinase inhibited activation of Akt that follows CD147 ligation, indicating that activation of PI3 Kinase is required for CD147 induced Akt activation. Similarly, c-Jun Kinase activation was greatly decreased by the PI3 Kinase inhibitor. Although the MEK inhibitor did not completely inhibit Erk1/2 activation, it greatly decreased c-Jun Kinase activation indicating that MEK is required for c-Jun Kinase activation downstream of CD147 ligation. Increased expression of CD147 has been suggested to result in enhanced survival of transformed cells. Together with our results of increased Akt, Erk and JNK phosphorylation in response to CD147 ligation, a theme of CD147 promoting a survival signal is emerging. These CD147 signal transduction studies indicate that tumor cell survival may be linked not only to induction of matrix metalloproteinases, but to downstream signaling through CD147 as well.
Proceedings of the National Academy of Sciences of the United States of America, Feb 1, 1989
Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its... more Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its physiological function is unknown, but in vitro studies suggest that Thy-1 may transmit activation signals to T cells and may play a role in the growth and/or differentiation of thymocytes [
Proceedings of the National Academy of Sciences of the United States of America, Apr 14, 1998
Differential expression of surface markers can frequently be used to distinguish functional subse... more Differential expression of surface markers can frequently be used to distinguish functional subsets of T cells, yet a surface phenotype unique to T cells induced into an anergic state has not been described. Here, we report that CD4 T cells rendered anergic in vivo by superantigen can be identified by loss of the 6C10 T cell marker. Inoculation of V8.1 T cell antigen receptor (TCR) transgenic mice with a V8.1-reactive minor lymphocyte-stimulating superantigen (Mls-1 a ) induces tolerance to Mls-1 a by clonal anergy. CD4 lymph node T cells from Mls-1 a inoculated transgenic mice enriched for the 6C10 ؊ phenotype neither proliferate nor produce interleukin-2 upon TCR engagement, whereas 6C10 ؉ CD4 T cells retain responsiveness. Analysis of T cell memory markers demonstrate that 6C10 ؊ T cells remain 3G11 hi but express heterogeneous levels of CD45RB, CD62L, CD44, and the CD69 early activation marker, suggesting that T cells at various degrees of activation can be functionally anergic. These studies demonstrate that anergic T cells can be purified based on 6C10 expression permitting examination of issues concerning biochemical and biological features specific to T cell anergy.
Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial... more Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.
This review is primarily concerned with unusual subsets of thymus-derived lymphocytes which our l... more This review is primarily concerned with unusual subsets of thymus-derived lymphocytes which our laboratory has analyzed. One of the many difficulties encountered in the study of T-cell differentiation pathways is that the actual numbers of T-lineage cells at a given stage of development is limited. Several methods have been conceived to overcome these difilculties, including establishment of precursor T-cell clones, thymocyte tumors or hybridomas, and microculture systems. Our laboratory has taken advantage of the gid mutation, which has dramatic effects on lymphocyte accumulation and positioning patterns. We will briefly discuss T-cell differentiation in general and then describe our experiments concerning novel T-cell subsets in peripheral lymph nodes of C3Hgld/gld mice. Finally, we will discuss our views concerning the origins of these novel T-ceil subsets and their relation to normal thymic differentiation.
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 1992
The bcI-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to ex... more The bcI-2 gene, which is overexpressed in human follicular B-cell lymphomas, has been found to extend ceflular lifespan through Inhibition of apoptosis, or pro- grammed cell death. However, the physiological role of the Bcd-2 protein In lymphocyte development is unclear. We have established a transgenic mouse line that expresses high levels of the Bcd-2 protein in both cortical and medulary thymocytes, disrupting the normal pattern of expression of this gene. We found that in these mice, immature thymocytes became resist- ant to apoptosis mediated by corticosterolds and calcium ionophores. Untreated thymocytes also exhibited a survival advantage in suspension cultures compared with controls. In addition, overexpression of bcI-2 enabled a proportion of thymocytes and peripheral T cells to escape the process of clonal deletion, which normally eliminates self-reactive T cells during thymocyte maturation. These findings implicate the Bcl-2 protein in regulating the lifespan of maturing thymocytes and in the antigenic-selection process.
Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxyge... more Thioredoxin peroxidase (TPx) is an antioxidant protein that limits the activity of reactive oxygen species (ROS). We cloned the cDNA encoding the mouse homolog of TPx from an E14.5 brain cDNA library and analyzed its distribution and function in murine tissues. Comparison of the amino acid sequence of mouse TPx with those of other species revealed that TPx was highly conserved across all species. Mouse TPx had broad tissue distribution, but its expression was especially marked in cells that metabolize oxygen molecules at high levels such as erythroid cells, renal tubular cells, cardiac and skeletal muscle cells, and certain types of neurons. Levels of increased expression of TPx in the brain were coincident with regions known to be especially sensitive to hypoxic and ischemic injury in humans. Models of erythroid differentiation and neuronal survival were employed to study the function of TPx. Murine erythroleukemia cells (MEL cells) increased TPx transcription when in a chemically differentiated state. Furthermore, expression of mouse TPx in PC12 pheochromocytoma cells prolonged their survival in the absence of nerve growth factor (NGF) and serum, indicating that TPx could promote neuronal cell survival. We propose that TPx contributes to antioxidant defense in erythrocytes and neuronal cells by limiting the destructive capacity of oxygen radicals. These findings identify a novel gene that appears to be relevant to hypoxic brain injury and may be of importance in development of new approaches to abrogate the effects of ischemic- and hypoxic-related injury in the central nervous system (CNS).
p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our rece... more p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (T reg ) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185 her2/neu (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit T reg functions in targeted therapies for cancers.
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Papers by Mark I Greene