Elena Loukinova - Academia.edu (original) (raw)
Papers by Elena Loukinova
Annals of the New York Academy of Sciences, Sep 1, 1994
Otolaryngology-Head and Neck Surgery, Aug 1, 1997
Journal of Cellular Biochemistry, 1997
Molecular changes occurring with tumor formation and metastasis need to be identified in order to... more Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin alpha was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-alpha family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets.
Clinical & Experimental Metastasis, Oct 1, 1998
Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at ... more Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at elevated levels in tissue homogenates, serum or supernatants of cell lines derived from patients with squamous cell carcinoma (SCC) [1-7], and other cancers [8-11]. Human and murine tumor lines often express the mRNA and secrete the proteins of these proinflammatory cytokines constitutively [1,5-7, 10-12]. Individual members of the proinflammatory
Journal of Biological Chemistry, Jul 1, 2005
Activation of the platelet-derived growth factor receptor- (PDGFR-) leads to tyrosine phosphory... more Activation of the platelet-derived growth factor receptor- (PDGFR-) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR- was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-, we confirmed that the kinase domain of PDGFR- is absolutely required for LRP tyrosine phosphorylation but that PDGFR--mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-␥, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR- may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR- coimmunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR- and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.
Oncogene, Jul 20, 2000
Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of ... more Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-a, designated mGRO-a, or KC. We have examined the possible role of mGRO-a expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 de®cient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-a and malignant potential with a pActin-KC vector encoding mGRO-a enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in in®ltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 de®cient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-a can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.
Otolaryngology-Head and Neck Surgery, Oct 1, 1998
Journal of Biological Chemistry, Jun 1, 1995
The FASEB Journal, Aug 1, 2000
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC... more High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE 2 production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1␣, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
Otolaryngology- Head and Neck Surgery, 1997
discharge patterns of bushy and stellate ceils, respectively, after deafferentation. The rectific... more discharge patterns of bushy and stellate ceils, respectively, after deafferentation. The rectification found in the currentvoltage relationship of bushy cells and the subthreshold linear relationship of stellate cells were also preserved. In both cell types the input resistance was significantly increased, and in bushy cells the resting membrane potential was significantly less negative after cochlear ablation. Compared with controls, deafferented stellate cells showed an increased action potential spike width at half height and decreased maximum rate of fall of the action potential, but these were not statistically significant. However, there was a statistically significant decrease in the ratio of pre-peak to postpeak spike width, and there was an increase in the ratio of maximum rates of rise to fall of the action potential. Taken together, these measures indicate that there is a delayed repolarization of the action potential. The increased input resistance and decreased resting ...
Journal of Biological Chemistry, May 3, 2002
Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein... more Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cellsurface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded 125 I-Fn at an increased rate, compared with MEF-2 cells.
Journal of Biological Chemistry
Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein... more Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length ...
Cancer research, Jan 15, 2001
To identify changes in gene expression with transformation and metastasis, we investigated differ... more To identify changes in gene expression with transformation and metastasis, we investigated differential gene expression in a squamous carcinoma model established in syngeneic mice. We used mRNA differential display (DD) to detect global differences and cDNA arrays enriched for cancer-associated genes using mRNA from primary keratinocytes, transformed Pam 212 squamous carcinoma cells, and metastases of Pam 212. After DD, 72 candidate cDNAs expressed primarily in transformed and metastatic cells were selected and cloned. Fifty-seven were detected, and 32 were confirmed to be differentially expressed by Northern blot analysis. mRNA expression profiles were also generated using a mouse cDNA array composed of 4000 elements representing known genes and expressed sequence tags plus the 57 DD candidate cDNAs detected by Northern analysis to facilitate data validation. cDNA array detected 76.9% of the differentially expressed mRNAs selected from DD and confirmed by Northern blot, whereas low...
The FASEB Journal, 2000
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC... more High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE 2 production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1␣, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
Oncogene, 2000
Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of ... more Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-a, designated mGRO-a, or KC. We have examined the possible role of mGRO-a expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 de®cient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-a and malignant potential with a pActin-KC vector encoding mGRO-a enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in in®ltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 de®cient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-a can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.
Journal of Cellular Biochemistry, 1997
Molecular changes occurring with tumor formation and metastasis need to be identified in order to... more Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin alpha was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-alpha family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets.
Journal of Biological Chemistry, 1995
International Journal of Cancer, 2001
We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in mu... more We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.
Clinical & Experimental Metastasis, 1998
Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at ... more Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at elevated levels in tissue homogenates, serum or supernatants of cell lines derived from patients with squamous cell carcinoma (SCC) [1-7], and other cancers [8-11]. Human and murine tumor lines often express the mRNA and secrete the proteins of these proinflammatory cytokines constitutively [1,5-7, 10-12]. Individual members of the proinflammatory
Annals of the New York Academy of Sciences, Sep 1, 1994
Otolaryngology-Head and Neck Surgery, Aug 1, 1997
Journal of Cellular Biochemistry, 1997
Molecular changes occurring with tumor formation and metastasis need to be identified in order to... more Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin alpha was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-alpha family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets.
Clinical & Experimental Metastasis, Oct 1, 1998
Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at ... more Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at elevated levels in tissue homogenates, serum or supernatants of cell lines derived from patients with squamous cell carcinoma (SCC) [1-7], and other cancers [8-11]. Human and murine tumor lines often express the mRNA and secrete the proteins of these proinflammatory cytokines constitutively [1,5-7, 10-12]. Individual members of the proinflammatory
Journal of Biological Chemistry, Jul 1, 2005
Activation of the platelet-derived growth factor receptor- (PDGFR-) leads to tyrosine phosphory... more Activation of the platelet-derived growth factor receptor- (PDGFR-) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR- was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-, we confirmed that the kinase domain of PDGFR- is absolutely required for LRP tyrosine phosphorylation but that PDGFR--mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-␥, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR- may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR- coimmunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR- and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.
Oncogene, Jul 20, 2000
Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of ... more Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-a, designated mGRO-a, or KC. We have examined the possible role of mGRO-a expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 de®cient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-a and malignant potential with a pActin-KC vector encoding mGRO-a enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in in®ltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 de®cient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-a can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.
Otolaryngology-Head and Neck Surgery, Oct 1, 1998
Journal of Biological Chemistry, Jun 1, 1995
The FASEB Journal, Aug 1, 2000
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC... more High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE 2 production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1␣, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
Otolaryngology- Head and Neck Surgery, 1997
discharge patterns of bushy and stellate ceils, respectively, after deafferentation. The rectific... more discharge patterns of bushy and stellate ceils, respectively, after deafferentation. The rectification found in the currentvoltage relationship of bushy cells and the subthreshold linear relationship of stellate cells were also preserved. In both cell types the input resistance was significantly increased, and in bushy cells the resting membrane potential was significantly less negative after cochlear ablation. Compared with controls, deafferented stellate cells showed an increased action potential spike width at half height and decreased maximum rate of fall of the action potential, but these were not statistically significant. However, there was a statistically significant decrease in the ratio of pre-peak to postpeak spike width, and there was an increase in the ratio of maximum rates of rise to fall of the action potential. Taken together, these measures indicate that there is a delayed repolarization of the action potential. The increased input resistance and decreased resting ...
Journal of Biological Chemistry, May 3, 2002
Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein... more Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cellsurface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded 125 I-Fn at an increased rate, compared with MEF-2 cells.
Journal of Biological Chemistry
Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein... more Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length ...
Cancer research, Jan 15, 2001
To identify changes in gene expression with transformation and metastasis, we investigated differ... more To identify changes in gene expression with transformation and metastasis, we investigated differential gene expression in a squamous carcinoma model established in syngeneic mice. We used mRNA differential display (DD) to detect global differences and cDNA arrays enriched for cancer-associated genes using mRNA from primary keratinocytes, transformed Pam 212 squamous carcinoma cells, and metastases of Pam 212. After DD, 72 candidate cDNAs expressed primarily in transformed and metastatic cells were selected and cloned. Fifty-seven were detected, and 32 were confirmed to be differentially expressed by Northern blot analysis. mRNA expression profiles were also generated using a mouse cDNA array composed of 4000 elements representing known genes and expressed sequence tags plus the 57 DD candidate cDNAs detected by Northern analysis to facilitate data validation. cDNA array detected 76.9% of the differentially expressed mRNAs selected from DD and confirmed by Northern blot, whereas low...
The FASEB Journal, 2000
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC... more High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE 2 production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1␣, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
Oncogene, 2000
Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of ... more Growth Regulated Oncogene-a (GRO-a) is an autocrine growth factor in melanoma and is a member of the C-X-C family of chemokines which promote chemotaxis of granulocytes and endothelia through binding to CXC Receptor 2. We found previously that variants of murine squamous cell carcinoma PAM 212 which grow and metastasize more rapidly in vivo constitutively express increased levels of murine GRO-a, designated mGRO-a, or KC. We have examined the possible role of mGRO-a expression in malignant progression of squamous cell carcinoma PAM 212 in homologous BALB/c and BALB CXC Receptor-2 de®cient mice. Transfection of the PAM 212 cell line which exhibits low expression of GRO-a and malignant potential with a pActin-KC vector encoding mGRO-a enabled isolation of PAM-KC expressing cell lines. These PAM-KC transfectants displayed an increased rate of growth and metastasis in BALB/c mice, similar to the highly malignant phenotype observed in spontaneously occurring metastatic variants. Furthermore, the PAM-KC tumors showed an increase in in®ltration of host leukocytes and CD31+ blood vessels, consistent with increased CXC chemokine activity. The increased growth of PAM-KC cells was attenuated in CXCR-2 de®cient mice, indicating that the increased growth was dependent in part upon host cells responsive to the CXC chemokine. Together, these results show that a CXC chemokine such as GRO-a can promote malignant growth of murine squamous cell carcinoma by a host CXCR-2 dependent pathway.
Journal of Cellular Biochemistry, 1997
Molecular changes occurring with tumor formation and metastasis need to be identified in order to... more Molecular changes occurring with tumor formation and metastasis need to be identified in order to define novel markers and targets for chemoprevention and therapy. Cell lines from a multistage model of murine squamous cell carcinoma were analyzed for differences in gene expression using mRNA differential display. mRNA was isolated from primary keratinocytes, an in vitro transformed keratinocyte line (Pam 212), and three metastatic cell lines derived from Pam 212 following tumor progression in vivo. cDNA was synthesized by reverse transcription and amplified by PCR using 72 primer combinations to screen and compare approximately 3,600 sequences. Five cDNAs with a differential expression pattern confirmed by Northern blot analysis were cloned and sequenced, revealing homology with known genes. The gene encoding tropomyosin alpha was preferentially expressed in primary keratinocytes; genes for tyrosine kinase Yes-associated protein (YAP65) and ribosomal protein L18a were preferentially expressed in transformed and metastatic tumor cell lines; and genes for the Gro-alpha family cytokine KC and antigen Sp17 exhibited increased expression in the three metastatic cell lines. The structure and function of the genes identified suggest that they may possibly be linked to cell shape and motility, signal transduction, protein synthesis, growth, granulocyte chemotaxis, and angiogenesis. This study demonstrates the ability of mRNA differential display to detect altered gene expression in this tumor progression model of murine squamous cell carcinoma, and the potential usefulness of this approach for identification of candidate genes as chemoprevention markers and targets.
Journal of Biological Chemistry, 1995
International Journal of Cancer, 2001
We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in mu... more We previously reported that chemokine Growth Regulated Oncogene 1 (Gro 1) is over-expressed in murine squamous cell carcinoma (SCC) with metastatic tumor progression. The enhanced expression of Gro-1 gene by SCC is regulated by activation of nuclear factor-kappaB (NF-kappaB), leading to accelerated tumor growth, angiogenesis and metastasis in vivo. In our study, we investigated the effect of the regulatory cytokines, IL-1alpha, EGF and TGF-beta1 on activation of NF-kappaB and Gro1 in primary and metastatic sublines of the murine SCC Pam 212. We found that Gro 1 expression could be induced by IL-1alpha or EGF in the low cytokine producing Pam 212 cells, but no significant induction was observed in high cytokine producing and metastatic LY-2 cells. Conditioned medium from LY-2 containing functional IL-1alpha induced Gro 1 expression in Pam 212 cells, which can be blocked by IL-1 receptor antagonist (IL-1RA). IL-1RA, however, had a minimal effect on constitutive Gro 1 production by LY-2 cells. TGF-beta1 suppressed constitutive as well as IL-1alpha and EGF-inducible Gro 1 production in both Pam 212 and LY-2 cells. IL-1alpha and EGF, but not TGF-beta1, were found to activate NF-kappaB in Pam 212, whereas none of the stimulants showed a significant effect on constitutive activation of NF-kappaB in LY-2 cells. Overexpression of a super repressor IkappaBalphaM in Pam 212 inhibited NF-kappaB binding activity, which led to impaired Gro 1 induction by IL-1alpha and EGF. These results demonstrate that IL-1alpha, EGF, and TGF-beta1 are important modulators of Gro 1 expression in SCC. Different responses to these modulators observed along with SCC metastatic progression may suggest a transition mechanism(s) for Gro 1 expression from host factor dependent to an independent stage involving NF-kappaB activation. Published 2001 Wiley-Liss, Inc.
Clinical & Experimental Metastasis, 1998
Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at ... more Several proinflammatory cytokines, including IL-1␣, IL-6, IL-8 and GM-CSF, have been detected at elevated levels in tissue homogenates, serum or supernatants of cell lines derived from patients with squamous cell carcinoma (SCC) [1-7], and other cancers [8-11]. Human and murine tumor lines often express the mRNA and secrete the proteins of these proinflammatory cytokines constitutively [1,5-7, 10-12]. Individual members of the proinflammatory