The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Justin J Mccormick - One of the best experts on this subject based on the ideXlab platform.
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transfection of a myc gene as a means of generating infinite life span human fibroblast Strains
Methods in Cell Science, 1995Co-Authors: Justin J Mccormick, Suzanne K Kohler, Veronica M MaherAbstract:Human fibroblasts in culture have never been found to transform spontaneously into immortal Cells. In an effort to generate an infinite life span Cell Strain from foreskin-derived normal diploid fibroblasts, we transfected the Cells with a plasmid carrying a v-myc oncogene linked to theneo gene, or with a control vector carrying theneo gene, and selected drug-resistant clones. A clone that expressed the v-myc protein was propagated to the end of its life span, with periodic cryogenic storage of the progeny. The population went into crisis at the same time as Cells from the control population and eventually senesced. However, while the Cells were senescing, viable-appearing clones were noted. The Cells of these clones continued to multiply, very slowly at first but eventually at a faster rate. Analysis showed that these Cells have a diploid karyotype that has remained stable throughout more than 200 population doublings since their sibling Cells senesced. Molecular analysis showed that the infinite life span Cells are, indeed, derived from the Cells used for transfection, and that they continue to express the v-myc protein.
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failure of infinite life span human Cells from different immortality complementation groups to yield finite life span hybrids
Journal of Cellular Physiology, 1994Co-Authors: Ann P Ryan, Veronica M Maher, Justin J MccormickAbstract:The observation that fusion of infinite life span Cells with finite life span Cells produces hybrid Cells with finite life spans led to the conclusion that an infinite life span in culture is a recessive trait resulting from loss of the function of a gene or genes that contribute to an active program for Cellular senescence. Furthermore, finding that certain pairs of infinite life span Cells, when fused to one another, can complement each other to yield finite life span hybrids allowed 30 infinite life span Cell lines to be assigned to four immortality complementation groups (Pereira-Smith and Smith, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:6042). In the present study, we fused a chromosomally stable, near diploid, morphologically normal, infinite life span Cell Strain, designated MSU-1.1, with its normal, finite life span, precursor Cell Strain and obtained finite life span hybrids, as expected if infinite life span in culture is a recessive trait. However, 14 of the 14 hybrids from our fusions of MSU-1.1 Cells with representative Cell lines from each of the four immortality complementation groups, and 38 of the 39 hybrids from our fusions of infinite life span Cells that have been reported to complement each other, failed to exhibit finite life spans. This result suggests that infinite life span Cells cannot complement each other to yield finite life span hybrids. In examining this unexpected result, we obtained evidence that long-term dual drug selection can be deleterious to hybrid Cells even though they carry resistance markers for both drugs, indicating that the Cell death of such hybrids observed in other studies may have resulted from the cytotoxic effect of long-term drug selection, rather than from senescence.
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characteristics of an infinite life span diploid human fibroblast Cell Strain and a near diploid Strain arising from a clone of Cells expressing a transfectedv myc oncogene
Experimental Cell Research, 1991Co-Authors: Thomas L Morgan, Veronica M Maher, Dennis G Fry, Dajun Yang, Peter J Hurlin, Suzanne K Kohler, Justin J MccormickAbstract:Abstract Diploid human fibroblasts were transfected with a plasmid carrying av- myc oncogene linked to the neo gene or with a vector control carrying a neo gene. Drug-resistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed thev- myc oncogene. After several months, these Cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This Cell Strain was designated MSU-1.1. Similar analysis showed that Cells from an earlier passage were diploid. These Cells were designated MSU-1.0. Both Strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both Strains express thev- myc protein and have the same integration site for the transfectedv- myc and neo genes. The MSU-1.0 Cells cannot grow without exogenously added growth factors. The MSU-1.1 Cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 Cells. Since the chance of human Cells acquiring an infinite life span in culture is very rare, the data suggest that MSU-1.1 Cells are derived from MSU-1.0 Cells. The expression ofv- myc is probably required for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression ofv- myc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 Cells expressed this oncogene, but the vast majority of them senesced.
Nitai P. Bhattacharyya - One of the best experts on this subject based on the ideXlab platform.
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resistance to induction of micronuclei chromosomal aberrations and apoptosis by 60co gamma ray in a Cell Strain m5 derived from chinese hamster v79 Cells
Journal of Radioanalytical and Nuclear Chemistry, 2007Co-Authors: Rupak Pathak, A Khuda R Bukhsh, Subrata Kumar Dey, Utpal Ghosh, Sen B Gupta, M Semwal, Nitai P. BhattacharyyaAbstract:The paper aims to investigate cytogenetic and apoptotic responses of γ-irradiation in a radio-resistant Cell Strain designated as M5. Induced micronuclei, chromosomal aberrations, nuclear fragmentation and nucleosomal ladders by γ-irradiation were less at equal doses in M5 Cells in comparison with that obtained in the parental Chinese hamster V79 Cells. However, at equal survival, there were no differences in the end points studied. Results indicate that the residual damages that lead to reproductive Cell death also resulted in the cytogenetic and apoptotic responses. We speculate that the repair efficiency in M5 Cells was more efficient and increased DNA repair could be the cause of radiation resistance observed in M5 Cells.
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increased expression of genes in a radioresistant Cell Strain modulation of hnrnp e2 hsp90 and ssbp2 genes in γ irradiated chinese hamster v79 Cells
International Journal of Low Radiation, 2007Co-Authors: Paromita Roychoudhury, Keya Chaudhuri, Bhaswati Pandit, Rupak Pathak, Nitai P. BhattacharyyaAbstract:The methotrexate-resistant Cell Strain M5, derived from Chinese hamster lung fibroblast Cell line V79, provides a good model to study differential gene expression in response to stress. Genes coding for human homologues of myosin phosphatase (AF458589), filamin-like protein (AAA92644), phosphate transporter 1 (AAH199441) and hnRNP E2 (NP_035172.1) were upregulated in M5 as determined by RAP-PCR. The Gene Expression Array (GEArray) analysis showed increased expression of caspase-6 and Hsp90 in M5 Cells compared to that observed in V79 Cells. Genomic amplification of SSBP2 as well as its transcriptional upregulation was also detected in M5 Cells. Following γ-irradiation, expressions of SSBP2, Hsp90 and hnRNP E2 increased steadily in V79 Cells. However, expression of hnRNP K and hnRNP A2 genes decreased at first and then increased, while hnRNP C1 gene expression remained unaffected up to the time studied. Thus, SSBP2, Hsp90 and hnRNP E2 are involved in the radiation response of V79 Cells.
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activation of mitochondrial promoter ph binding protein in a radio resistant chinese hamster Cell Strain associated with bcl 2
Biochemical and Biophysical Research Communications, 2006Co-Authors: Paromita Roychoudhury, Utpal Ghosh, Nitai P. Bhattacharyya, Keya ChaudhuriAbstract:The Cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P(H)) has been increased in a radio-resistant Cell Strain designated as M5 in comparison with the parental Chinese hamster V79 Cells. These genes are also up regulated in Chinese hamster V79 Cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P(L)) was found to be similar in both the Cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial Cell extracts from these two Cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 Cell extracts interacted with the P(H) promoter in comparison to that observed with Cell extracts from Chinese hamster V79 Cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 Cells was due to enhanced interaction proteins with the promoters P(H) and mediated by the expression of Bcl2.
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Identification of two differentially expressed mitochondrial genes in a methotrexate-resistant Chinese hamster Cell Strain derived from V79 Cells using RNA fingerprinting by arbitrary primed polymerase chain reaction
Radiation research, 2003Co-Authors: Keya Chaudhuri, Ramanuj Banerjee, Bhaswati Pandit, Aparna Mukherjee, Soumita Das, Sharmila Sengupta, Susanta Roychoudury, Nitai P. BhattacharyyaAbstract:Abstract Chaudhuri, K., Banerjee, R., Pandit, B., Mukherjee, A., Das, S., Sengupta, S., Roychoudury, S. and Bhattacharyya, N. P. Identification of Two Differentially Expressed Mitochondrial Genes in a Methotrexate-Resistant Chinese Hamster Cell Strain Derived from V79 Cells Using RNA Fingerprinting by Arbitrary Primed Polymerase Chain Reaction. Radiat. Res. 160, 77–85 (2003). To identify genes that are differentially expressed in a methotrexate (MTX)-resistant Cell Strain designated as M5 that exhibits resistance to γ radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 Cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was ab...
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possible role of glutathione in resistance to heavy metals and hydrogen peroxide in a radioresistant chinese hamster v79 Cell Strain
Indian Journal of Experimental Biology, 1996Co-Authors: Sharmila Sengupta, Nitai P. BhattacharyyaAbstract:To understand the Cellular and biochemical nature of radioresistance in the Strain M5 derived from Chinese hamster V79 Cells, the sensitivity of the resistant Cells towards CdCl2, Zn(Ac)2, and H2O2 by the colony forming ability has been tested. D0 values for these compounds in Chinese hamster V79 Cells were 5.4 microM, 27.8 microM and 4.3 micrograms/ml respectively while for M5 Cells these were 8.3 microM, 142.9 microM and 11.9 micrograms/ml respectively. The resistance to heavy metals as well as the oxidative damage could be reversed by the inhibition of glutathione synthesis by the drug buthionine sulfoximine (BSO). These set of data indicate that the Cellular antioxidant glutathione plays an important role in the observed oxidant-resistant phenotype as well as heavy metal resistance in M5 Cells.
Keya Chaudhuri - One of the best experts on this subject based on the ideXlab platform.
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increased expression of genes in a radioresistant Cell Strain modulation of hnrnp e2 hsp90 and ssbp2 genes in γ irradiated chinese hamster v79 Cells
International Journal of Low Radiation, 2007Co-Authors: Paromita Roychoudhury, Keya Chaudhuri, Bhaswati Pandit, Rupak Pathak, Nitai P. BhattacharyyaAbstract:The methotrexate-resistant Cell Strain M5, derived from Chinese hamster lung fibroblast Cell line V79, provides a good model to study differential gene expression in response to stress. Genes coding for human homologues of myosin phosphatase (AF458589), filamin-like protein (AAA92644), phosphate transporter 1 (AAH199441) and hnRNP E2 (NP_035172.1) were upregulated in M5 as determined by RAP-PCR. The Gene Expression Array (GEArray) analysis showed increased expression of caspase-6 and Hsp90 in M5 Cells compared to that observed in V79 Cells. Genomic amplification of SSBP2 as well as its transcriptional upregulation was also detected in M5 Cells. Following γ-irradiation, expressions of SSBP2, Hsp90 and hnRNP E2 increased steadily in V79 Cells. However, expression of hnRNP K and hnRNP A2 genes decreased at first and then increased, while hnRNP C1 gene expression remained unaffected up to the time studied. Thus, SSBP2, Hsp90 and hnRNP E2 are involved in the radiation response of V79 Cells.
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activation of mitochondrial promoter ph binding protein in a radio resistant chinese hamster Cell Strain associated with bcl 2
Biochemical and Biophysical Research Communications, 2006Co-Authors: Paromita Roychoudhury, Utpal Ghosh, Nitai P. Bhattacharyya, Keya ChaudhuriAbstract:The Cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P(H)) has been increased in a radio-resistant Cell Strain designated as M5 in comparison with the parental Chinese hamster V79 Cells. These genes are also up regulated in Chinese hamster V79 Cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P(L)) was found to be similar in both the Cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial Cell extracts from these two Cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 Cell extracts interacted with the P(H) promoter in comparison to that observed with Cell extracts from Chinese hamster V79 Cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 Cells was due to enhanced interaction proteins with the promoters P(H) and mediated by the expression of Bcl2.
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Identification of two differentially expressed mitochondrial genes in a methotrexate-resistant Chinese hamster Cell Strain derived from V79 Cells using RNA fingerprinting by arbitrary primed polymerase chain reaction
Radiation research, 2003Co-Authors: Keya Chaudhuri, Ramanuj Banerjee, Bhaswati Pandit, Aparna Mukherjee, Soumita Das, Sharmila Sengupta, Susanta Roychoudury, Nitai P. BhattacharyyaAbstract:Abstract Chaudhuri, K., Banerjee, R., Pandit, B., Mukherjee, A., Das, S., Sengupta, S., Roychoudury, S. and Bhattacharyya, N. P. Identification of Two Differentially Expressed Mitochondrial Genes in a Methotrexate-Resistant Chinese Hamster Cell Strain Derived from V79 Cells Using RNA Fingerprinting by Arbitrary Primed Polymerase Chain Reaction. Radiat. Res. 160, 77–85 (2003). To identify genes that are differentially expressed in a methotrexate (MTX)-resistant Cell Strain designated as M5 that exhibits resistance to γ radiation and a number of chemotherapeutic drugs compared to the parental Chinese hamster V79 Cells, we used RNA fingerprinting by arbitrary primed polymerase chain reaction (RAP-PCR). By comparative analysis, we identified six differentially expressed transcripts that were cloned and sequenced. Two of these partial cDNA clones showed high homology to the mitochondrial genes NADH dehydrogenase subunit 1 and subunit 4. The steady-state mRNA level of both the NADH dehydrogenase subunits was ab...
Veronica M Maher - One of the best experts on this subject based on the ideXlab platform.
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transfection of a myc gene as a means of generating infinite life span human fibroblast Strains
Methods in Cell Science, 1995Co-Authors: Justin J Mccormick, Suzanne K Kohler, Veronica M MaherAbstract:Human fibroblasts in culture have never been found to transform spontaneously into immortal Cells. In an effort to generate an infinite life span Cell Strain from foreskin-derived normal diploid fibroblasts, we transfected the Cells with a plasmid carrying a v-myc oncogene linked to theneo gene, or with a control vector carrying theneo gene, and selected drug-resistant clones. A clone that expressed the v-myc protein was propagated to the end of its life span, with periodic cryogenic storage of the progeny. The population went into crisis at the same time as Cells from the control population and eventually senesced. However, while the Cells were senescing, viable-appearing clones were noted. The Cells of these clones continued to multiply, very slowly at first but eventually at a faster rate. Analysis showed that these Cells have a diploid karyotype that has remained stable throughout more than 200 population doublings since their sibling Cells senesced. Molecular analysis showed that the infinite life span Cells are, indeed, derived from the Cells used for transfection, and that they continue to express the v-myc protein.
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failure of infinite life span human Cells from different immortality complementation groups to yield finite life span hybrids
Journal of Cellular Physiology, 1994Co-Authors: Ann P Ryan, Veronica M Maher, Justin J MccormickAbstract:The observation that fusion of infinite life span Cells with finite life span Cells produces hybrid Cells with finite life spans led to the conclusion that an infinite life span in culture is a recessive trait resulting from loss of the function of a gene or genes that contribute to an active program for Cellular senescence. Furthermore, finding that certain pairs of infinite life span Cells, when fused to one another, can complement each other to yield finite life span hybrids allowed 30 infinite life span Cell lines to be assigned to four immortality complementation groups (Pereira-Smith and Smith, 1988, Proc. Natl. Acad. Sci. U.S.A., 85:6042). In the present study, we fused a chromosomally stable, near diploid, morphologically normal, infinite life span Cell Strain, designated MSU-1.1, with its normal, finite life span, precursor Cell Strain and obtained finite life span hybrids, as expected if infinite life span in culture is a recessive trait. However, 14 of the 14 hybrids from our fusions of MSU-1.1 Cells with representative Cell lines from each of the four immortality complementation groups, and 38 of the 39 hybrids from our fusions of infinite life span Cells that have been reported to complement each other, failed to exhibit finite life spans. This result suggests that infinite life span Cells cannot complement each other to yield finite life span hybrids. In examining this unexpected result, we obtained evidence that long-term dual drug selection can be deleterious to hybrid Cells even though they carry resistance markers for both drugs, indicating that the Cell death of such hybrids observed in other studies may have resulted from the cytotoxic effect of long-term drug selection, rather than from senescence.
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characteristics of an infinite life span diploid human fibroblast Cell Strain and a near diploid Strain arising from a clone of Cells expressing a transfectedv myc oncogene
Experimental Cell Research, 1991Co-Authors: Thomas L Morgan, Veronica M Maher, Dennis G Fry, Dajun Yang, Peter J Hurlin, Suzanne K Kohler, Justin J MccormickAbstract:Abstract Diploid human fibroblasts were transfected with a plasmid carrying av- myc oncogene linked to the neo gene or with a vector control carrying a neo gene. Drug-resistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed thev- myc oncogene. After several months, these Cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This Cell Strain was designated MSU-1.1. Similar analysis showed that Cells from an earlier passage were diploid. These Cells were designated MSU-1.0. Both Strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both Strains express thev- myc protein and have the same integration site for the transfectedv- myc and neo genes. The MSU-1.0 Cells cannot grow without exogenously added growth factors. The MSU-1.1 Cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 Cells. Since the chance of human Cells acquiring an infinite life span in culture is very rare, the data suggest that MSU-1.1 Cells are derived from MSU-1.0 Cells. The expression ofv- myc is probably required for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression ofv- myc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 Cells expressed this oncogene, but the vast majority of them senesced.
Thomas J Moehring - One of the best experts on this subject based on the ideXlab platform.
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structural and functional analysis of the protein products derived from mutant fur alleles in an endoprotease deficient chinese hamster ovary Cell Strain
Somatic Cell and Molecular Genetics, 1998Co-Authors: Joseph F Sucic, Michael J Spence, Thomas J MoehringAbstract:The fur gene encodes the endoprotease, furin. We recently demonstrated mutations in both fur alleles in the mutant Chinese hamster ovary (CHO)-K1 Strain, RPE.40, and hypothesized that these mutations were responsible for the endoprotease-deficient phenotype of these Cells. We now present the structural and functional properties of three protein products derived from the mutant fur alleles. None of these protein products were able to process the precursor to von Willebrand factor, which is processed by wild-type furin. Pro-protein processing activity initially attributed to one of the mutant proteins was due to wild-type furin produced inadvertently from one of the expression constructs used in these experiments. None of the mutant proteins exhibited evidence of autocatalysis, consistent with the lack of activity versus the test substrate, and glycosylation patterns suggested at least two of them remained in the endoplasmic reticulum. These results confirm that RPE.40 Cells are furin null mutants, as earlier evidence had suggested.
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analysis of mutations in alleles of the fur gene from an endoprotease deficient chinese hamster ovary Cell Strain
Somatic Cell and Molecular Genetics, 1995Co-Authors: Michael J Spence, Joseph F Sucic, Brian T Foley, Thomas J MoehringAbstract:RPE. 40 mutant Cells differ from wild-type Chinese hamster ovary (CHO-K1) Cells in their increased resistance toPseudomonas exotoxin A and their inability to process the insulin proreceptor and certain viral envelope proproteins. Northern analysis revealed that RPE. 40 Cells maintained a substantially lower steady-state level of 4.0 kbfur mRNA than did CHO-K1 Cells. Analysis offur cDNAs showed that RPE. 40 Cells were diploid at thefur locus, and RPE. 40 Cells had a Cys (TGC) to Tyr (TAC) mutation in codon 196 of one allele (allele I). Approximately 25–30% of the CHO-K1 Cells were also heterozygous (Tyr/Cys) at codon 196, and pre-mRNAs transcribed from the second allele (allele II) in RPE. 40 Cells were defectively spliced. All other pre-mRNAs were correctly spliced. Rapid turnover of defectively spliced transcripts may account for the reduced steady-state level offur mRNA observed in RPE. 40 Cells. Our results provide a mechanistic basis for the endoprotease-deficient phenotype of RPE. 40 Cells.
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defective processing of the insulin receptor in an endoprotease deficient chinese hamster Cell Strain is corrected by expression of mouse furin
Journal of Biological Chemistry, 1993Co-Authors: B J Robertson, J M Moehring, Thomas J MoehringAbstract:Abstract Characterization of an endoprotease-deficient mutant Chinese hamster ovary (CHO) Cell, designated RPE.40, revealed that it bound less than 10% as much insulin as did its parent, CHO-K1. We examined processing of the endogenous insulin receptor in CHO-K1 and RPE.40 Cells, and processing of the human insulin receptor expressed in these Cells. RPE.40 Cells did not process the endogenous insulin proreceptor to its subunit forms, and processed the human insulin proreceptor inefficiently. Accumulation of the proreceptor form of the insulin receptor was seen in both cases. Furin is a mammalian endoprotease that cleaves proproteins at a consensus sequence of basic amino acids found in the insulin proreceptor. Expression of mouse furin in RPE.40 Cells restored normal processing of the endogenous and the human insulin receptor in these Cells. In addition, expression of mouse furin corrected the reduced binding of insulin in RPE.40 Cells, indicating that receptor function as well as processing was restored.