The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A ... more The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reducÃ-asewas higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 /IM) caused an 85-90% depression of HMG CoA reducÃ-aseactivity and of the incorporation of [•'Hjacetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 /IM) also abolished essentially all dolichol synthesis, as measured by incorporation of |3H|acetate. In con trast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reducÃ-ase, the cellular growth as well as dolichol synthesis was significantly decreased. Since the inhibitory effect of 25-OH on HMG CoA reducÃ-aseactivily did noi exceed lhat of mevinolin, it seems that 25-OH, besides inhibiting HMG CoA reducÃ-ase, inhibils sleps disiai lo HMG CoA reducÃ-ase. This nolion was further supported by Ihe finding lhal addition of mevalonale did noi prevenl Ihe 25-OH-induced growth inhibition. However, if dolichol was added along wilh 25-OH, Ihe block was partially prevenled, indicating lhal a critical level of de novo synlhesis of dolichol is essential for cellular growlh.
Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme ... more Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.
The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of gen... more The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression ...
The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A ... more The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 microM) caused an 85-90% depression of HMG CoA reductase activity and of the incorporation of [3H]acetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 microM) also abolished essentially all dilichol synthesis, as measured by incorporation of [3H]acetate. In contrast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reducta...
Many studies have noted an association between amplification of single oncogenes and poor prognos... more Many studies have noted an association between amplification of single oncogenes and poor prognosis in breast cancer. We are investigating whether measurement of amplification in a larger number of proto-oncogenes increases the reliability of the prognostic information provided. As the first stage of this investigation, amplification (of c-erbB-2, cycD1, int-2, c-myc and MDM2), aneuploidy and altered expression of p53, which all indicate genetic instability, were studied in 117 primary breast adenocarcinomas. Amplification was correlated with aneuploidy (p=0.002) but not with altered expression of p53 even though the tumours with p53 overexpression were all aneuploid. Our results suggest that measurement of amplification is a potentially valuable prognostic factor.
The restriction point (R) is defined as the point in G 1 after which cells can complete a divisio... more The restriction point (R) is defined as the point in G 1 after which cells can complete a division cycle without growth factors and divides G 1 into two physiologically different intervals in cycling cells, G 1 -pm (a postmitotic interval with a constant length of 3 to 4 h) and G 1 -ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G 1 regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G 1 cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.
The restriction point (R) separates the G1 phase of continuously cycling cells into two functiona... more The restriction point (R) separates the G1 phase of continuously cycling cells into two functionally different parts. The first part, G1-pm, represents the growth factor dependent post-mitotic interval from mitosis to R, which is of constant length (3-4 h). The second part, G1-ps, represents the growth factor independent, pre-S phase interval of G1 that lasts from R to S and that varies in time from 1 to 10 h. G1-pm cells rapidly exit (within 1 h) from the cell cycle and enter G0 as a response to serum withdrawal. The finding that R occurs at a set time after mitosis indicates that R may be related to the metabolic and/or structural changes that the cell underwent during the previous mitosis. We have recently shown that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is not the molecular mechanism behind R, as has been suggested previously. Here, we present an alternative explanation for R. In the present study, we applied a single cell approach using time-lapse analysis, which revealed that upon serum starvation the G1-pm cells rapidly underwent a transient change in cell shape from flat to spherical before exiting to G0. Platelet derived growth factor (PDGF) counteracted this change in shape and also prevented exit to G0 to the same extent. Furthermore epidermal growth factor (EGF) and insulin like growth factor (IGF-1), which only partially counteracted this change, only partially counteracts exit to G0. These data clearly indicate a direct link between change in cell shape and exit to G0 in G1-cells that have not passed R.
Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme ... more Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.
In this study we investigated whether early G1 cells can be collected by the method of centrifuga... more In this study we investigated whether early G1 cells can be collected by the method of centrifugal elutriation, and in particular whether the stringent growth regulatory properties characteristic of early G1 cells really are preserved in elutriated cells. We compared the cell cycle kinetics of elutriated Swiss 3T3 cells with unperturbed cells which had been analyzed by time-lapse video recording. Our data show that centrifugal elutriation yields a good synchronization of early G1 cells. However, from a strict cell cycle kinetic point of view the elutriated cells preserve their growth regulatory properties to a variable extent. This fact was explained by the finding that the early G1 cells tend to undergo a temporary G0 arrest as a result of the elutriation procedures.
Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erb B-2 at 17... more Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erb B-2 at 17q11.2 ف q21.1 is frequently amplified, as is a region at 17q22 ف q24. As a step in the search for the target gene(s) of the 17q22-q24 amplification we determined whether the placental lactogen (PL) genes at 17q23 were amplified in 59 breast carcinomas. These genes were selected as their upregulation could theoretically be involved in breast cancer tumorigenesis. Amplification of the PL genes, and also of cerb B-2, was detected using semi-quantitative PCR. The reliability of this method was confirmed since c-erb B-2 results obtained using PCR, Southern blotting and immunohistochemistry were in good agreement. The PL genes were amplified in 13 (22%) of the tumors. Furthermore, the PL and cerb B-2 genes were frequently co-amplified although there is a nonamplified region between them. Expression of PL was investigated in 26 tumors and was detected in 16 of these cases including all 10 tumors with amplification of the PL genes. The tumors with PL gene amplification were all aneuploid. A trend was seen towards an increased incidence of lymph node involvement for tumors with amplification of the PL genes and for tumors with co-amplification of PL and c-erb B-2, which suggests a possible association with high malignancy.
Biochemical and Biophysical Research Communications, 1996
Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involv... more Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involved in many cellular processes. Protein phosphatase 5 (PP5) is a novel member of the protein serine/threonine phosphatase family. The majority of the cDNA sequence of PP5 has been reported recently. In our study, a sequence encoding the whole open reading frame of PP5 was cloned from a human fetal brain cDNA library. The protein phosphatase cDNA sequence of our clone is longer at the 5Ј end than the recently published sequence. It's likely that the extended sequence contains the start codon ATG, since a translation stop codon TAG is present upstream of the ATG codon in the same open reading frame. The mRNA of the PP5 gene was detected in all the human tissues examined. The PP5 gene was localized to human chromosomal region 19q13.3.
Background: Germline mutations in the folliculin (FLCN) gene are associated with the development ... more Background: Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied. Methods: BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets.
The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A ... more The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reducÃ-asewas higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 /IM) caused an 85-90% depression of HMG CoA reducÃ-aseactivity and of the incorporation of [•'Hjacetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 /IM) also abolished essentially all dolichol synthesis, as measured by incorporation of |3H|acetate. In con trast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reducÃ-ase, the cellular growth as well as dolichol synthesis was significantly decreased. Since the inhibitory effect of 25-OH on HMG CoA reducÃ-aseactivily did noi exceed lhat of mevinolin, it seems that 25-OH, besides inhibiting HMG CoA reducÃ-ase, inhibils sleps disiai lo HMG CoA reducÃ-ase. This nolion was further supported by Ihe finding lhal addition of mevalonale did noi prevenl Ihe 25-OH-induced growth inhibition. However, if dolichol was added along wilh 25-OH, Ihe block was partially prevenled, indicating lhal a critical level of de novo synlhesis of dolichol is essential for cellular growlh.
Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme ... more Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.
The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of gen... more The study of kidney cancer pathogenesis and its treatment has been limited by the scarcity of genetically defined animal models. The FLCN gene that codes for the protein folliculin, mutated in Birt-Hogg-Dubé syndrome, presents a new target for mouse modeling of kidney cancer. Here we developed a kidney-specific knockout model by disrupting the mouse Flcn in the proximal tubules, thus avoiding homozygous embryonic lethality or neonatal mortality, and eliminating the requirement of loss of heterozygosity for tumorigenesis. This knockout develops renal cysts and early onset (6 months) of multiple histological subtypes of renal neoplasms featuring high tumor penetrance. Although the majority of the tumors were chromophobe renal cell carcinomas in affected mice under 1 year of age, papillary renal cell carcinomas predominated in the kidneys of older knockout mice. This renal neoplasia from cystic hyperplasia at 4 months to high-grade renal tumors by 16 months represented the progression ...
The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A ... more The basal level of the gene expression and the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was higher in SV40-transformed human fibroblasts (90-VA VI) than in normal ones (HDF). In both these cell types mevinolin (25 microM) caused an 85-90% depression of HMG CoA reductase activity and of the incorporation of [3H]acetate into sterols. In HDF this was coupled to an efficient block of cell growth, whereas the growth of 90-VA VI was only slightly reduced by mevinolin. In HDF, mevinolin (25 microM) also abolished essentially all dilichol synthesis, as measured by incorporation of [3H]acetate. In contrast, dolichol synthesis remained unaltered, or was increased, in mevinolin-treated 90-VA VI. We suggest that these different responses of dolichol synthesis may depend on different substrate affinities of the rate-limiting enzyme in the dolichol pathway. However, if 90-VA VI was treated with 25-hydroxycholesterol (25-OH), an alternative inhibitor of HMG CoA reducta...
Many studies have noted an association between amplification of single oncogenes and poor prognos... more Many studies have noted an association between amplification of single oncogenes and poor prognosis in breast cancer. We are investigating whether measurement of amplification in a larger number of proto-oncogenes increases the reliability of the prognostic information provided. As the first stage of this investigation, amplification (of c-erbB-2, cycD1, int-2, c-myc and MDM2), aneuploidy and altered expression of p53, which all indicate genetic instability, were studied in 117 primary breast adenocarcinomas. Amplification was correlated with aneuploidy (p=0.002) but not with altered expression of p53 even though the tumours with p53 overexpression were all aneuploid. Our results suggest that measurement of amplification is a potentially valuable prognostic factor.
The restriction point (R) is defined as the point in G 1 after which cells can complete a divisio... more The restriction point (R) is defined as the point in G 1 after which cells can complete a division cycle without growth factors and divides G 1 into two physiologically different intervals in cycling cells, G 1 -pm (a postmitotic interval with a constant length of 3 to 4 h) and G 1 -ps (a pre-DNA-synthetic interval with a variable length of 1 to 10 h). Cyclin E is a G 1 regulatory protein whose accumulation has been suggested to be critical for passage through R. We have studied cyclin E protein levels in individual cells of asynchronously growing cell populations, with respect to both passage through R and entry into S phase. We found that the postmitotic G 1 cells that had not yet reached R were negative for cyclin E accumulation. On the other hand, cells that had passed R were found to accumulate cyclin E at variable times (1 to 8 h) after passage through R and 2 to 5 h before entry into S. These kinetic data rule out the hypothesis that passage through R is dependent on the accumulation of cyclin E but suggest, instead, the converse, that passage through R is a prerequisite for cyclin E accumulation. Furthermore, we found that most of the cyclin E protein is downregulated within 1 to 2 h after entry into S.
The restriction point (R) separates the G1 phase of continuously cycling cells into two functiona... more The restriction point (R) separates the G1 phase of continuously cycling cells into two functionally different parts. The first part, G1-pm, represents the growth factor dependent post-mitotic interval from mitosis to R, which is of constant length (3-4 h). The second part, G1-ps, represents the growth factor independent, pre-S phase interval of G1 that lasts from R to S and that varies in time from 1 to 10 h. G1-pm cells rapidly exit (within 1 h) from the cell cycle and enter G0 as a response to serum withdrawal. The finding that R occurs at a set time after mitosis indicates that R may be related to the metabolic and/or structural changes that the cell underwent during the previous mitosis. We have recently shown that phosphorylation of the retinoblastoma tumor suppressor protein (pRb) is not the molecular mechanism behind R, as has been suggested previously. Here, we present an alternative explanation for R. In the present study, we applied a single cell approach using time-lapse analysis, which revealed that upon serum starvation the G1-pm cells rapidly underwent a transient change in cell shape from flat to spherical before exiting to G0. Platelet derived growth factor (PDGF) counteracted this change in shape and also prevented exit to G0 to the same extent. Furthermore epidermal growth factor (EGF) and insulin like growth factor (IGF-1), which only partially counteracted this change, only partially counteracts exit to G0. These data clearly indicate a direct link between change in cell shape and exit to G0 in G1-cells that have not passed R.
Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme ... more Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.
In this study we investigated whether early G1 cells can be collected by the method of centrifuga... more In this study we investigated whether early G1 cells can be collected by the method of centrifugal elutriation, and in particular whether the stringent growth regulatory properties characteristic of early G1 cells really are preserved in elutriated cells. We compared the cell cycle kinetics of elutriated Swiss 3T3 cells with unperturbed cells which had been analyzed by time-lapse video recording. Our data show that centrifugal elutriation yields a good synchronization of early G1 cells. However, from a strict cell cycle kinetic point of view the elutriated cells preserve their growth regulatory properties to a variable extent. This fact was explained by the finding that the early G1 cells tend to undergo a temporary G0 arrest as a result of the elutriation procedures.
Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erb B-2 at 17... more Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erb B-2 at 17q11.2 ف q21.1 is frequently amplified, as is a region at 17q22 ف q24. As a step in the search for the target gene(s) of the 17q22-q24 amplification we determined whether the placental lactogen (PL) genes at 17q23 were amplified in 59 breast carcinomas. These genes were selected as their upregulation could theoretically be involved in breast cancer tumorigenesis. Amplification of the PL genes, and also of cerb B-2, was detected using semi-quantitative PCR. The reliability of this method was confirmed since c-erb B-2 results obtained using PCR, Southern blotting and immunohistochemistry were in good agreement. The PL genes were amplified in 13 (22%) of the tumors. Furthermore, the PL and cerb B-2 genes were frequently co-amplified although there is a nonamplified region between them. Expression of PL was investigated in 26 tumors and was detected in 16 of these cases including all 10 tumors with amplification of the PL genes. The tumors with PL gene amplification were all aneuploid. A trend was seen towards an increased incidence of lymph node involvement for tumors with amplification of the PL genes and for tumors with co-amplification of PL and c-erb B-2, which suggests a possible association with high malignancy.
Biochemical and Biophysical Research Communications, 1996
Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involv... more Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involved in many cellular processes. Protein phosphatase 5 (PP5) is a novel member of the protein serine/threonine phosphatase family. The majority of the cDNA sequence of PP5 has been reported recently. In our study, a sequence encoding the whole open reading frame of PP5 was cloned from a human fetal brain cDNA library. The protein phosphatase cDNA sequence of our clone is longer at the 5Ј end than the recently published sequence. It's likely that the extended sequence contains the start codon ATG, since a translation stop codon TAG is present upstream of the ATG codon in the same open reading frame. The mRNA of the PP5 gene was detected in all the human tissues examined. The PP5 gene was localized to human chromosomal region 19q13.3.
Background: Germline mutations in the folliculin (FLCN) gene are associated with the development ... more Background: Germline mutations in the folliculin (FLCN) gene are associated with the development of Birt-Hogg-Dubé syndrome (BHDS), a disease characterized by papular skin lesions, a high occurrence of spontaneous pneumothorax, and the development of renal neoplasias. The majority of renal tumors that arise in BHDS-affected individuals are histologically similar to sporadic chromophobe renal cell carcinoma (RCC) and sporadic renal oncocytoma. However, most sporadic tumors lack FLCN mutations and the extent to which the BHDS-derived renal tumors share genetic defects associated with the sporadic tumors has not been well studied. Methods: BHDS individuals were identified symptomatically and FLCN mutations were confirmed by DNA sequencing. Comparative gene expression profiling analyses were carried out on renal tumors isolated from individuals afflicted with BHDS and a panel of sporadic renal tumors of different subtypes using discriminate and clustering approaches. qRT-PCR was used to confirm selected results of the gene expression analyses. We further analyzed differentially expressed genes using gene set enrichment analysis and pathway analysis approaches. Pathway analysis results were confirmed by generation of independent pathway signatures and application to additional datasets.
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Papers by Peter Zickert