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Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy.

Yu W, Kim J, Ossowski L - J. Cell Biol. (1997)

Bottom Line: Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls.In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness.This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo-passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These "reemerged," uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro-grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.

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uPAR-mRNA level in cells isolated from reestablished  CAM tumors. (A) CAM tumors were dissociated into single-cell  suspension, plated, and kept in culture for 1–2 wk. Total RNA  was extracted from ∼1 × 107 cells and analyzed by Northern blot  hybridization exactly as described in the legend to Fig. 1. Film was  exposed for 40 min after hybridization with labeled uPAR-cDNA  and for 20 min after GAPDH probes. (B) Units calculated from a  densitometry scan and expressed as described in the legend to  Fig. 1. Only clones AS 32 and 36 had detectable antisense RNA,  both when grown in culture and after forming CAM tumors. Circled numbers indicate cells after in vivo growth.
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Figure 5: uPAR-mRNA level in cells isolated from reestablished CAM tumors. (A) CAM tumors were dissociated into single-cell suspension, plated, and kept in culture for 1–2 wk. Total RNA was extracted from ∼1 × 107 cells and analyzed by Northern blot hybridization exactly as described in the legend to Fig. 1. Film was exposed for 40 min after hybridization with labeled uPAR-cDNA and for 20 min after GAPDH probes. (B) Units calculated from a densitometry scan and expressed as described in the legend to Fig. 1. Only clones AS 32 and 36 had detectable antisense RNA, both when grown in culture and after forming CAM tumors. Circled numbers indicate cells after in vivo growth.

Mentions: We conclude that low uPAR expression coincides with a dramatically lengthened period of cancer dormancy. As noticed from Fig. 4, although dormancy was sustained for a long time period, it was not permanent since at 4 to 5 mo all uPAR-deficient clones regained the ability to grow in vivo (Fig. 4). Was regrowth linked to reexpression of uPAR? To answer this, uPAR-mRNA level and pro-uPA binding were measured in tumors that emerged from dormancy by dissociating and growing the cells for 1 wk in culture. Fig. 5, A and B, and Table I show that uPAR both at the mRNA and receptor number levels remained low.


Reduction in surface urokinase receptor forces malignant cells into a protracted state of dormancy.

Yu W, Kim J, Ossowski L - J. Cell Biol. (1997)

uPAR-mRNA level in cells isolated from reestablished  CAM tumors. (A) CAM tumors were dissociated into single-cell  suspension, plated, and kept in culture for 1–2 wk. Total RNA  was extracted from ∼1 × 107 cells and analyzed by Northern blot  hybridization exactly as described in the legend to Fig. 1. Film was  exposed for 40 min after hybridization with labeled uPAR-cDNA  and for 20 min after GAPDH probes. (B) Units calculated from a  densitometry scan and expressed as described in the legend to  Fig. 1. Only clones AS 32 and 36 had detectable antisense RNA,  both when grown in culture and after forming CAM tumors. Circled numbers indicate cells after in vivo growth.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139877&req=5

Figure 5: uPAR-mRNA level in cells isolated from reestablished CAM tumors. (A) CAM tumors were dissociated into single-cell suspension, plated, and kept in culture for 1–2 wk. Total RNA was extracted from ∼1 × 107 cells and analyzed by Northern blot hybridization exactly as described in the legend to Fig. 1. Film was exposed for 40 min after hybridization with labeled uPAR-cDNA and for 20 min after GAPDH probes. (B) Units calculated from a densitometry scan and expressed as described in the legend to Fig. 1. Only clones AS 32 and 36 had detectable antisense RNA, both when grown in culture and after forming CAM tumors. Circled numbers indicate cells after in vivo growth.
Mentions: We conclude that low uPAR expression coincides with a dramatically lengthened period of cancer dormancy. As noticed from Fig. 4, although dormancy was sustained for a long time period, it was not permanent since at 4 to 5 mo all uPAR-deficient clones regained the ability to grow in vivo (Fig. 4). Was regrowth linked to reexpression of uPAR? To answer this, uPAR-mRNA level and pro-uPA binding were measured in tumors that emerged from dormancy by dissociating and growing the cells for 1 wk in culture. Fig. 5, A and B, and Table I show that uPAR both at the mRNA and receptor number levels remained low.

Bottom Line: Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls.In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness.This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the beta-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo-passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These "reemerged," uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro-grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.

Show MeSH
Related in: MedlinePlus