Nonneoplastic cell lines are unable to grow in soft agar. However, concomitant treatment of these... more Nonneoplastic cell lines are unable to grow in soft agar. However, concomitant treatment of these cells with epidermal growth factor and transforming growth factor beta confers upon them anchorage independence. Since articular chondrocytes are unique as normal diploid cells that do have the capability of growing in soft agar, we tested whether transforming growth factor beta and epidermal growth factor could affect DNA synthesis and matrix production. In the presence of epidermal growth factor (5 ng/ml) concentrations of high-performance liquid chromatography-purified transforming growth factor beta at concentrations of 0.05-15 ng/ml induced a dose-dependent increase in DNA, to nearly double that of control cultures. A half-maximal effect was seen with transforming growth factor beta, 0.1 ng/ml, and epidermal growth factor, 5 ng/ml. Neither compound alone was mitogenic. In contrast, either transforming growth factor beta or epidermal growth factor alone was able to decrease synthesi...
Proceedings of the National Academy of Sciences, 1984
To facilitate our studies on the mechanisms controlling collagenase production at a molecular lev... more To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The inc...
Proceedings of the National Academy of Sciences, 1985
Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein... more Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein that prevented the synthesis of the neutral proteinase collagenase. Conditioned medium was concentrated 10-fold and placed on cultures of rabbit synovial fibroblasts along with an inducer of collagenase (phorbol myristate acetate or latex particles) and [3H]leucine. Collagenase production was measured by immunoprecipitation of culture medium with monospecific antibody. Gel filtration showed that the inhibitory factor had MrS of 12,500, 25,000-50,000, and 150,000, suggesting that the protein may exist as aggregates. Activity was destroyed by boiling, by trypsin, and by dithiothreitol. Production of the inhibitory protein was prevented by cycloheximide. Isoelectric focusing purified the protein 100- to 150-fold and revealed pIs in the range of 3.2-3.7. Glycosylation was demonstrated by binding to Con A-Sepharose. Our data indicate that rabbit synovial fibroblasts autoregulate collagenase ...
Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer dea... more Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF(V600E), results in constitutive activation of the mitogen-activated protein kinase pathway. In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF(V600E) affected cytokine (IL-1β, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts. We found that BRAF(V600E) melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF(V600E) melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma ce...
Summary Autoantibodies to pancreas, ileum, kidney and liver extracts were formed in the sera of t... more Summary Autoantibodies to pancreas, ileum, kidney and liver extracts were formed in the sera of three of eighteen rabbits immunized with pooled rabbit pancreas extract. The autoantibodies were shown to be present in the serum prior to immunization, although injection of pooled rabbit pancreas extract with complete Freund's adjuvant increased their titer. Absorption with any autologous organ extract removed reactivity to all other autologous extracts, while absorption with allogeneic ileum or kidney removed activity to these extracts, but not to pancrease. Therefore, along with increased production of autoantibody, pancreas-specific isoantibodies were formed. However, no pancreas-specific autoantibodies were found following immunization with pooled rabbit pancreas extract. Sucrose density gradient ultracentrifugation showed autoantibody in both 19 S and 7 S globulins. Heating the antisera to 65°C for 30 min destroyed their complement-fixing ability.
Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with cle... more Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with clear cell RCC arising from mutations in the von Hippel Lindau (VHL) tumor suppressor. Successful RCC tumor suppression by VHL requires the negative regulation of hypoxia inducible factor alpha (HIF alpha) protein and its downstream targets. Thus, identification of HIF target genes responsible for RCC tumor progression will aid in the development of therapies for this disease. We previously identified membrane type-1 matrix metalloproteinase (MT1-MMP) as a transcriptional target of HIF-2alpha in RCC cells null for VHL and showed that MT1-MMP is overexpressed in these cells. MT1-MMP is a key regulator of tumor progression through its functions as a matrix-degrading enzyme, as well as its ability to cleave factors, such as adhesion molecules and other MMPs. The aim of this study was to investigate the contribution of MT1-MMP to the invasive potential of RCC cells using in vitro type I collage...
The 1G/2G polymorphism of matrix metalloproteinase 1 (MMP-1) affects activity of the promoter in ... more The 1G/2G polymorphism of matrix metalloproteinase 1 (MMP-1) affects activity of the promoter in transient transfections, and has been associated with the incidence or invasiveness of five types of cancer. In light of these findings, and because stromal cells may contribute to tumor cell invasion, we used quantitative real-time reverse transcription-PCR to measure endogenous MMP-1 mRNA expression in 34 human foreskin fibroblasts homozygous or heterozygous for the 1G and 2G alleles. We measured basal, cytokine, and growth factor induced MMP-1 mRNA expression. The genotype of the MMP-1 promoter polymorphism was not predictive of mean MMP-1 mRNA expression. However, within the population of cell lines with at least one 2G polymorphism, there were more individuals with higher levels of MMP-1 mRNA after treatment with a cytokine or growth factors. Our data suggest that the presence of the 2G polymorphism does not significantly affect mean expression levels of a population but may increas...
Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabol... more Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabolism as the only enzyme capable of degrading interstitial collagens at neutral pH. We used fragments of the rabbit collagenase promoter ranging from 1800 to 182 bp to measure transcriptional activity of the activator protein-1 (AP-1) site at -77. Mutation at -77 in this sequence greatly reduced basal transcription in all constructs. However, mutant constructs with at least 321 bp of promoter responded to phorbol myristate acetate, similar to their native counterparts, implicating upstream regions in mediating this response. Through mutagenesis and analysis of DNA-protein interactions, we also identified and characterized a novel AP-1 site at -186. Mutation at -186 in 321 bp of promoter modestly lowered basal activity but, in contrast to mutation at -77, reduced phorbol responsiveness by 50%. Mobility shift assays demonstrated specific inducible binding at both sites. DNA/protein complexes at both AP-1 sites contain c-Fos and Jun D proteins, while Fra-2 is present only at the -77 site. These studies (1) demonstrate cooperativity between these two AP-1 sites, (2) implicate the -186 site in phorbol inducibility and (3) identify specific members of the Fos and Jun families binding to these sites.
Nonneoplastic cell lines are unable to grow in soft agar. However, concomitant treatment of these... more Nonneoplastic cell lines are unable to grow in soft agar. However, concomitant treatment of these cells with epidermal growth factor and transforming growth factor beta confers upon them anchorage independence. Since articular chondrocytes are unique as normal diploid cells that do have the capability of growing in soft agar, we tested whether transforming growth factor beta and epidermal growth factor could affect DNA synthesis and matrix production. In the presence of epidermal growth factor (5 ng/ml) concentrations of high-performance liquid chromatography-purified transforming growth factor beta at concentrations of 0.05-15 ng/ml induced a dose-dependent increase in DNA, to nearly double that of control cultures. A half-maximal effect was seen with transforming growth factor beta, 0.1 ng/ml, and epidermal growth factor, 5 ng/ml. Neither compound alone was mitogenic. In contrast, either transforming growth factor beta or epidermal growth factor alone was able to decrease synthesi...
Proceedings of the National Academy of Sciences, 1984
To facilitate our studies on the mechanisms controlling collagenase production at a molecular lev... more To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The inc...
Proceedings of the National Academy of Sciences, 1985
Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein... more Conditioned medium taken from cultures of resting rabbit synovial fibroblasts contained a protein that prevented the synthesis of the neutral proteinase collagenase. Conditioned medium was concentrated 10-fold and placed on cultures of rabbit synovial fibroblasts along with an inducer of collagenase (phorbol myristate acetate or latex particles) and [3H]leucine. Collagenase production was measured by immunoprecipitation of culture medium with monospecific antibody. Gel filtration showed that the inhibitory factor had MrS of 12,500, 25,000-50,000, and 150,000, suggesting that the protein may exist as aggregates. Activity was destroyed by boiling, by trypsin, and by dithiothreitol. Production of the inhibitory protein was prevented by cycloheximide. Isoelectric focusing purified the protein 100- to 150-fold and revealed pIs in the range of 3.2-3.7. Glycosylation was demonstrated by binding to Con A-Sepharose. Our data indicate that rabbit synovial fibroblasts autoregulate collagenase ...
Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer dea... more Melanoma, the most lethal form of skin cancer, is responsible for over 80% of all skin cancer deaths and is highly metastatic, readily spreading to the lymph nodes or metastasising to other organs. The frequent genetic mutation found in metastatic melanoma, BRAF(V600E), results in constitutive activation of the mitogen-activated protein kinase pathway. In this study, we utilised genetically engineered melanoma cell lines and xenograft mouse models to investigate how BRAF(V600E) affected cytokine (IL-1β, IL-6, and IL-8) and matrix metalloproteinase-1 (MMP-1) expression in tumour cells and in human dermal fibroblasts. We found that BRAF(V600E) melanoma cells expressed higher levels of these cytokines and of MMP-1 than wild-type counterparts. Further, conditioned medium from the BRAF(V600E) melanoma cells promoted the activation of stromal fibroblasts, inducing expression of SDF-1 and its receptor CXCR4. This increase was mitigated when the conditioned medium was taken from melanoma ce...
Summary Autoantibodies to pancreas, ileum, kidney and liver extracts were formed in the sera of t... more Summary Autoantibodies to pancreas, ileum, kidney and liver extracts were formed in the sera of three of eighteen rabbits immunized with pooled rabbit pancreas extract. The autoantibodies were shown to be present in the serum prior to immunization, although injection of pooled rabbit pancreas extract with complete Freund's adjuvant increased their titer. Absorption with any autologous organ extract removed reactivity to all other autologous extracts, while absorption with allogeneic ileum or kidney removed activity to these extracts, but not to pancrease. Therefore, along with increased production of autoantibody, pancreas-specific isoantibodies were formed. However, no pancreas-specific autoantibodies were found following immunization with pooled rabbit pancreas extract. Sucrose density gradient ultracentrifugation showed autoantibody in both 19 S and 7 S globulins. Heating the antisera to 65°C for 30 min destroyed their complement-fixing ability.
Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with cle... more Metastatic renal cell carcinoma (RCC) remains the leading cause of mortality in patients with clear cell RCC arising from mutations in the von Hippel Lindau (VHL) tumor suppressor. Successful RCC tumor suppression by VHL requires the negative regulation of hypoxia inducible factor alpha (HIF alpha) protein and its downstream targets. Thus, identification of HIF target genes responsible for RCC tumor progression will aid in the development of therapies for this disease. We previously identified membrane type-1 matrix metalloproteinase (MT1-MMP) as a transcriptional target of HIF-2alpha in RCC cells null for VHL and showed that MT1-MMP is overexpressed in these cells. MT1-MMP is a key regulator of tumor progression through its functions as a matrix-degrading enzyme, as well as its ability to cleave factors, such as adhesion molecules and other MMPs. The aim of this study was to investigate the contribution of MT1-MMP to the invasive potential of RCC cells using in vitro type I collage...
The 1G/2G polymorphism of matrix metalloproteinase 1 (MMP-1) affects activity of the promoter in ... more The 1G/2G polymorphism of matrix metalloproteinase 1 (MMP-1) affects activity of the promoter in transient transfections, and has been associated with the incidence or invasiveness of five types of cancer. In light of these findings, and because stromal cells may contribute to tumor cell invasion, we used quantitative real-time reverse transcription-PCR to measure endogenous MMP-1 mRNA expression in 34 human foreskin fibroblasts homozygous or heterozygous for the 1G and 2G alleles. We measured basal, cytokine, and growth factor induced MMP-1 mRNA expression. The genotype of the MMP-1 promoter polymorphism was not predictive of mean MMP-1 mRNA expression. However, within the population of cell lines with at least one 2G polymorphism, there were more individuals with higher levels of MMP-1 mRNA after treatment with a cytokine or growth factors. Our data suggest that the presence of the 2G polymorphism does not significantly affect mean expression levels of a population but may increas...
Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabol... more Collagenase (matrix metalloproteinase-1, MMP-1) plays a central role in connective tissue metabolism as the only enzyme capable of degrading interstitial collagens at neutral pH. We used fragments of the rabbit collagenase promoter ranging from 1800 to 182 bp to measure transcriptional activity of the activator protein-1 (AP-1) site at -77. Mutation at -77 in this sequence greatly reduced basal transcription in all constructs. However, mutant constructs with at least 321 bp of promoter responded to phorbol myristate acetate, similar to their native counterparts, implicating upstream regions in mediating this response. Through mutagenesis and analysis of DNA-protein interactions, we also identified and characterized a novel AP-1 site at -186. Mutation at -186 in 321 bp of promoter modestly lowered basal activity but, in contrast to mutation at -77, reduced phorbol responsiveness by 50%. Mobility shift assays demonstrated specific inducible binding at both sites. DNA/protein complexes at both AP-1 sites contain c-Fos and Jun D proteins, while Fra-2 is present only at the -77 site. These studies (1) demonstrate cooperativity between these two AP-1 sites, (2) implicate the -186 site in phorbol inducibility and (3) identify specific members of the Fos and Jun families binding to these sites.
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Papers by Constance Brinckerhoff