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Transdifferentiation-inducing HCCR-1 oncogene.

Ha SA, Kim HK, Yoo J, Kim S, Shin SM, Lee YS, Hur SY, Kim YW, Kim TE, Chung YJ, Jeun SS, Kim DW, Park YG, Kim J, Shin SY, Lee YH, Kim JW - BMC Cell Biol. (2010)

Bottom Line: This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis.Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney.Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, Catholic Medical Research Institute, The Catholic University of Korea, Seoul, Korea.

ABSTRACT

Background: Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.

Results: We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.

Conclusions: Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

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Related in: MedlinePlus

Molecular alterations in HCCR-1-induced tumorigenesis. A. Cell-cycle profiles of wild-type NIH/3T3 cell and HCCR-1-transfected cells from a separate experiment. Exponentially growing cells were trypsinized and DNA content was determined by flow cytometry. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% BCS for 36 houes. After incubation, cells were released with 20% serum and harvested. B. Tumor suppressor egr-1 expressions. HCCR-1-transfected cells were serum starved with 0.5% BCS for 36 hours, and then stimulated with fresh 20% serum. Total RNA was isolated, transferred, and hybridized with 32P-labeled egr-1, c-fos, and GAPDH probe, respectively. C. Determination of telomerase activity. Human telomerase-positive embryonic kidney 293 cells, 293 cell extracts treated with RNase (+ RNase), wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cells were analyzed by Telomerase PCR ELISA. Assays were performed according to the kit protocol with amounts of extracts equivalent to 1 ×10 3 cells. The telomerase activity in 293 cells, which served as a positive control, was abolished by pretreatment with RNase. Results are the average mean absorbance values from four separate experiments (means and 95% confidence intervals) (wild-type versus HCCR-1, P < .05). D. Determination of PKC activity. Wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cell extracts were prepared and assayed to determine PKC activity. Each value is the means and 95% confidence intervals of three independent experiments (wild-type versus HCCR-1, P < 0.05).
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Figure 3: Molecular alterations in HCCR-1-induced tumorigenesis. A. Cell-cycle profiles of wild-type NIH/3T3 cell and HCCR-1-transfected cells from a separate experiment. Exponentially growing cells were trypsinized and DNA content was determined by flow cytometry. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% BCS for 36 houes. After incubation, cells were released with 20% serum and harvested. B. Tumor suppressor egr-1 expressions. HCCR-1-transfected cells were serum starved with 0.5% BCS for 36 hours, and then stimulated with fresh 20% serum. Total RNA was isolated, transferred, and hybridized with 32P-labeled egr-1, c-fos, and GAPDH probe, respectively. C. Determination of telomerase activity. Human telomerase-positive embryonic kidney 293 cells, 293 cell extracts treated with RNase (+ RNase), wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cells were analyzed by Telomerase PCR ELISA. Assays were performed according to the kit protocol with amounts of extracts equivalent to 1 ×10 3 cells. The telomerase activity in 293 cells, which served as a positive control, was abolished by pretreatment with RNase. Results are the average mean absorbance values from four separate experiments (means and 95% confidence intervals) (wild-type versus HCCR-1, P < .05). D. Determination of PKC activity. Wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cell extracts were prepared and assayed to determine PKC activity. Each value is the means and 95% confidence intervals of three independent experiments (wild-type versus HCCR-1, P < 0.05).

Mentions: In order to study whether there was an alteration in the growth properties of HCCR-1-transfected cells, we examined cell cycle profiles. The percentage of wild-type NIH/3T3 cells and HCCR-1-transfected cells in the S-phase was 20.6% and 31.5%, respectively (Figure 3A, mid-log phase). These results suggest that there was a significant shift of the cell population out of the G 0/G 1-phase into the S-phase in HCCR-1-transfected cells. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% bovine calf serum (BCS) for 36 hours. After incubation, cells were released with 20% serum and harvested at the indicated times. In wild-type cells (measured at 0 hour), few cells remained in the S-phase (8%). In contrast, a considerable number of HCCR-1-transfected cells were still in the S-phase (21.8%), suggesting that constitutive overexpression of HCCR-1 allowed for a relative amount of resistance to serum deprivation-induced G 0/G 1 arrest. Following the release of cells from the growth arrest caused by serum-deprivation, there were consistent increases of over 10% in the S-phase populations of HCCR-1-transfected cells as compared to wild-type cells at measured time intervals (24 hour and 48 hour, respectively). Therefore, overexpression of HCCR-1 could deregulate cell growth by shortening the G 0/G 1-phase and increasing the S-phase population of cells. It has been implicated that egr-1 functions as a tumor-suppressor [24]. To assess whether HCCR-1-induced tumor formation is associated with the loss of egr-1 expression, we tested the time course of egr-1 expression. When quiescent cells were stimulated with 20% serum, a marked down-regulation of egr-1 was observed in HCCR-1-transfected cells compared with wild-type NIH/3T3 cells (Figure 3B). In contrast, upregulation of GAPDH mRNA level in HCCR-1-transfected cells was clearly seen, while no significant difference was observed in the level of c-fos (Figure 3B). This result suggests that down-regulation of tumor suppressor egr-1 may be involved in the tumor progression in HCCR-1-overexpressing cells. To further explain the tumorigenesis of HCCR-1, we determined the telomerase activity in PKC-activated HCCR-1-transfected cells. Consistent with a previous study [25],wild-type NIH/3T3 cells showed detectable telomerase activity (Figure 3C). However, HCCR-1 gene transfection increased telomerase activity up to about 7-fold when compared with wild-type cells. Reports show that PKC induces a marked increase in telomerase activity [26]. To determine whether the increased telomerase activity in HCCR-1 transfected cells is caused by PKC, a kinase assay was performed. PKC activity of HCCR-1-transfected cells was increased by about 10-fold when compared with wild-type cells (Figure 3D).


Transdifferentiation-inducing HCCR-1 oncogene.

Ha SA, Kim HK, Yoo J, Kim S, Shin SM, Lee YS, Hur SY, Kim YW, Kim TE, Chung YJ, Jeun SS, Kim DW, Park YG, Kim J, Shin SY, Lee YH, Kim JW - BMC Cell Biol. (2010)

Molecular alterations in HCCR-1-induced tumorigenesis. A. Cell-cycle profiles of wild-type NIH/3T3 cell and HCCR-1-transfected cells from a separate experiment. Exponentially growing cells were trypsinized and DNA content was determined by flow cytometry. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% BCS for 36 houes. After incubation, cells were released with 20% serum and harvested. B. Tumor suppressor egr-1 expressions. HCCR-1-transfected cells were serum starved with 0.5% BCS for 36 hours, and then stimulated with fresh 20% serum. Total RNA was isolated, transferred, and hybridized with 32P-labeled egr-1, c-fos, and GAPDH probe, respectively. C. Determination of telomerase activity. Human telomerase-positive embryonic kidney 293 cells, 293 cell extracts treated with RNase (+ RNase), wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cells were analyzed by Telomerase PCR ELISA. Assays were performed according to the kit protocol with amounts of extracts equivalent to 1 ×10 3 cells. The telomerase activity in 293 cells, which served as a positive control, was abolished by pretreatment with RNase. Results are the average mean absorbance values from four separate experiments (means and 95% confidence intervals) (wild-type versus HCCR-1, P < .05). D. Determination of PKC activity. Wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cell extracts were prepared and assayed to determine PKC activity. Each value is the means and 95% confidence intervals of three independent experiments (wild-type versus HCCR-1, P < 0.05).
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Figure 3: Molecular alterations in HCCR-1-induced tumorigenesis. A. Cell-cycle profiles of wild-type NIH/3T3 cell and HCCR-1-transfected cells from a separate experiment. Exponentially growing cells were trypsinized and DNA content was determined by flow cytometry. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% BCS for 36 houes. After incubation, cells were released with 20% serum and harvested. B. Tumor suppressor egr-1 expressions. HCCR-1-transfected cells were serum starved with 0.5% BCS for 36 hours, and then stimulated with fresh 20% serum. Total RNA was isolated, transferred, and hybridized with 32P-labeled egr-1, c-fos, and GAPDH probe, respectively. C. Determination of telomerase activity. Human telomerase-positive embryonic kidney 293 cells, 293 cell extracts treated with RNase (+ RNase), wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cells were analyzed by Telomerase PCR ELISA. Assays were performed according to the kit protocol with amounts of extracts equivalent to 1 ×10 3 cells. The telomerase activity in 293 cells, which served as a positive control, was abolished by pretreatment with RNase. Results are the average mean absorbance values from four separate experiments (means and 95% confidence intervals) (wild-type versus HCCR-1, P < .05). D. Determination of PKC activity. Wild-type NIH/3T3, cells transfected with vector alone and HCCR-1-transfected cell extracts were prepared and assayed to determine PKC activity. Each value is the means and 95% confidence intervals of three independent experiments (wild-type versus HCCR-1, P < 0.05).
Mentions: In order to study whether there was an alteration in the growth properties of HCCR-1-transfected cells, we examined cell cycle profiles. The percentage of wild-type NIH/3T3 cells and HCCR-1-transfected cells in the S-phase was 20.6% and 31.5%, respectively (Figure 3A, mid-log phase). These results suggest that there was a significant shift of the cell population out of the G 0/G 1-phase into the S-phase in HCCR-1-transfected cells. To assess the serum-dependent cell cycle progression, cells were cultured in 0.5% bovine calf serum (BCS) for 36 hours. After incubation, cells were released with 20% serum and harvested at the indicated times. In wild-type cells (measured at 0 hour), few cells remained in the S-phase (8%). In contrast, a considerable number of HCCR-1-transfected cells were still in the S-phase (21.8%), suggesting that constitutive overexpression of HCCR-1 allowed for a relative amount of resistance to serum deprivation-induced G 0/G 1 arrest. Following the release of cells from the growth arrest caused by serum-deprivation, there were consistent increases of over 10% in the S-phase populations of HCCR-1-transfected cells as compared to wild-type cells at measured time intervals (24 hour and 48 hour, respectively). Therefore, overexpression of HCCR-1 could deregulate cell growth by shortening the G 0/G 1-phase and increasing the S-phase population of cells. It has been implicated that egr-1 functions as a tumor-suppressor [24]. To assess whether HCCR-1-induced tumor formation is associated with the loss of egr-1 expression, we tested the time course of egr-1 expression. When quiescent cells were stimulated with 20% serum, a marked down-regulation of egr-1 was observed in HCCR-1-transfected cells compared with wild-type NIH/3T3 cells (Figure 3B). In contrast, upregulation of GAPDH mRNA level in HCCR-1-transfected cells was clearly seen, while no significant difference was observed in the level of c-fos (Figure 3B). This result suggests that down-regulation of tumor suppressor egr-1 may be involved in the tumor progression in HCCR-1-overexpressing cells. To further explain the tumorigenesis of HCCR-1, we determined the telomerase activity in PKC-activated HCCR-1-transfected cells. Consistent with a previous study [25],wild-type NIH/3T3 cells showed detectable telomerase activity (Figure 3C). However, HCCR-1 gene transfection increased telomerase activity up to about 7-fold when compared with wild-type cells. Reports show that PKC induces a marked increase in telomerase activity [26]. To determine whether the increased telomerase activity in HCCR-1 transfected cells is caused by PKC, a kinase assay was performed. PKC activity of HCCR-1-transfected cells was increased by about 10-fold when compared with wild-type cells (Figure 3D).

Bottom Line: This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis.Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney.Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetic Laboratory, Catholic Medical Research Institute, The Catholic University of Korea, Seoul, Korea.

ABSTRACT

Background: Cell transdifferentiation is characterized by loss of some phenotypes along with acquisition of new phenotypes in differentiated cells. The differentiated state of a given cell is not irreversible. It depends on the up- and downregulation exerted by specific molecules.

Results: We report here that HCCR-1, previously shown to play an oncogenic role in human cancers, induces epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) in human and mouse, respectively. The stem cell factor receptor CD117/c-Kit was induced in this transdifferentiated (EMT) sarcoma tissues. This MET occurring in HCCR-1 transfected cells is reminiscent of the transdifferentiation process during nephrogenesis. Indeed, expression of HCCR-1 was observed during the embryonic development of the kidney. This suggests that HCCR-1 might be involved in the transdifferentiation process of cancer stem cell.

Conclusions: Therefore, we propose that HCCR-1 may be a regulatory factor that stimulates morphogenesis of epithelia or mesenchyme during neoplastic transformation.

Show MeSH
Related in: MedlinePlus