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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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Reduction of uPAR-mRNA level in antisense-transfected clones. uPAR-mRNA steady-state levels were determined  in parental HEp3 cells, two control clones, and five uPAR antisense clones by Northern blot hybridization (30 μg total RNA)  using uPAR-cDNA (exposure 6 h) and, after stripping, GAPDHcDNA (exposure 1.5 h) as probes. The film was scanned, and the  values were expressed as units of uPAR-mRNA per unit of  GAPDH-mRNA and calculated as percent of uPAR-mRNA in  parental cells.
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Figure 1: Reduction of uPAR-mRNA level in antisense-transfected clones. uPAR-mRNA steady-state levels were determined in parental HEp3 cells, two control clones, and five uPAR antisense clones by Northern blot hybridization (30 μg total RNA) using uPAR-cDNA (exposure 6 h) and, after stripping, GAPDHcDNA (exposure 1.5 h) as probes. The film was scanned, and the values were expressed as units of uPAR-mRNA per unit of GAPDH-mRNA and calculated as percent of uPAR-mRNA in parental cells.

Mentions: The levels of uPAR-mRNA were determined by Northern blotting and expressed as uPAR-mRNA units per unit of GAPDH-mRNA. The uPAR-mRNA in parental HEp3 cells was taken as 100%. Fig. 1 shows that the reduced uPAR number was coincidental with a reduced (51 to 80%) uPAR-mRNA level in the antisense clones and that the relatively constant level of mRNA in the two control clones tested was comparable to that in the parental HEp3 cells. Only two clones (AS 32 and 36) had easily detectable antisense RNA bands (results not shown). We and others have shown that reduction in mRNA level of the targeted gene is not always proportional to the level of detectable antisense RNA (van der Krol et al., 1988; Kasid et al., 1989; Neckers et al., 1992; Kook et al., 1994). The reduction in uPAR remained 50% or better in all the antisense clones after at least 4 mo in culture (Tables I and II).


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

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

Reduction of uPAR-mRNA level in antisense-transfected clones. uPAR-mRNA steady-state levels were determined  in parental HEp3 cells, two control clones, and five uPAR antisense clones by Northern blot hybridization (30 μg total RNA)  using uPAR-cDNA (exposure 6 h) and, after stripping, GAPDHcDNA (exposure 1.5 h) as probes. The film was scanned, and the  values were expressed as units of uPAR-mRNA per unit of  GAPDH-mRNA and calculated as percent of uPAR-mRNA in  parental cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Reduction of uPAR-mRNA level in antisense-transfected clones. uPAR-mRNA steady-state levels were determined in parental HEp3 cells, two control clones, and five uPAR antisense clones by Northern blot hybridization (30 μg total RNA) using uPAR-cDNA (exposure 6 h) and, after stripping, GAPDHcDNA (exposure 1.5 h) as probes. The film was scanned, and the values were expressed as units of uPAR-mRNA per unit of GAPDH-mRNA and calculated as percent of uPAR-mRNA in parental cells.
Mentions: The levels of uPAR-mRNA were determined by Northern blotting and expressed as uPAR-mRNA units per unit of GAPDH-mRNA. The uPAR-mRNA in parental HEp3 cells was taken as 100%. Fig. 1 shows that the reduced uPAR number was coincidental with a reduced (51 to 80%) uPAR-mRNA level in the antisense clones and that the relatively constant level of mRNA in the two control clones tested was comparable to that in the parental HEp3 cells. Only two clones (AS 32 and 36) had easily detectable antisense RNA bands (results not shown). We and others have shown that reduction in mRNA level of the targeted gene is not always proportional to the level of detectable antisense RNA (van der Krol et al., 1988; Kasid et al., 1989; Neckers et al., 1992; Kook et al., 1994). The reduction in uPAR remained 50% or better in all the antisense clones after at least 4 mo in culture (Tables I and II).

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