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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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Summary of in  vitro and in vivo properties  of control and uPAR antisense clones. Cells of control  clones rapidly form large tumor masses on CAMs (large  open spheres), while cells in  which uPAR is inhibited remain dormant in vivo (small  circles) for as long as 5 mo  and then reemerge to form  tumors. However, while the  tumorigenic and metastatic  properties of the control  clones are stable, the compensatory mechanism operating in vivo and leading to  the interruption of dormancy  produces heterogeneous and  unstable phenotype that  never reaches the full malignant potential of the controls. H, high; I, intermediate; L, low.
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Figure 8: Summary of in vitro and in vivo properties of control and uPAR antisense clones. Cells of control clones rapidly form large tumor masses on CAMs (large open spheres), while cells in which uPAR is inhibited remain dormant in vivo (small circles) for as long as 5 mo and then reemerge to form tumors. However, while the tumorigenic and metastatic properties of the control clones are stable, the compensatory mechanism operating in vivo and leading to the interruption of dormancy produces heterogeneous and unstable phenotype that never reaches the full malignant potential of the controls. H, high; I, intermediate; L, low.

Mentions: The results of this study demonstrate that to initiate progressive tumor growth in vivo, epidermoid carcinoma (HEp3) cells require a full complement of surface uPAR. This conclusion is based on the observation, summarized in Fig. 8, that all control cells (parental and four independent control clones transfected with vector alone) with similarly high uPAR numbers produced actively growing tumors when inoculated on CAMs. In contrast, all five of the antisense clones, with receptor levels reduced by more than 50%, remained dormant for as long as 5 mo in vivo (Fig. 4). Two additional antisense clones (AS 3 and 26), in which uPAR was reduced by only 30%, emerged from dormancy after 6 and 7 wk in vivo, respectively (results not shown). This high degree of correlation between uPAR level and tumorigenicity or dormancy, exhibited by 11 independent clones, establishes a basis for proposing a novel function for uPAR.


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

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

Summary of in  vitro and in vivo properties  of control and uPAR antisense clones. Cells of control  clones rapidly form large tumor masses on CAMs (large  open spheres), while cells in  which uPAR is inhibited remain dormant in vivo (small  circles) for as long as 5 mo  and then reemerge to form  tumors. However, while the  tumorigenic and metastatic  properties of the control  clones are stable, the compensatory mechanism operating in vivo and leading to  the interruption of dormancy  produces heterogeneous and  unstable phenotype that  never reaches the full malignant potential of the controls. H, high; I, intermediate; L, low.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Summary of in vitro and in vivo properties of control and uPAR antisense clones. Cells of control clones rapidly form large tumor masses on CAMs (large open spheres), while cells in which uPAR is inhibited remain dormant in vivo (small circles) for as long as 5 mo and then reemerge to form tumors. However, while the tumorigenic and metastatic properties of the control clones are stable, the compensatory mechanism operating in vivo and leading to the interruption of dormancy produces heterogeneous and unstable phenotype that never reaches the full malignant potential of the controls. H, high; I, intermediate; L, low.
Mentions: The results of this study demonstrate that to initiate progressive tumor growth in vivo, epidermoid carcinoma (HEp3) cells require a full complement of surface uPAR. This conclusion is based on the observation, summarized in Fig. 8, that all control cells (parental and four independent control clones transfected with vector alone) with similarly high uPAR numbers produced actively growing tumors when inoculated on CAMs. In contrast, all five of the antisense clones, with receptor levels reduced by more than 50%, remained dormant for as long as 5 mo in vivo (Fig. 4). Two additional antisense clones (AS 3 and 26), in which uPAR was reduced by only 30%, emerged from dormancy after 6 and 7 wk in vivo, respectively (results not shown). This high degree of correlation between uPAR level and tumorigenicity or dormancy, exhibited by 11 independent clones, establishes a basis for proposing a novel function for uPAR.

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