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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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Invasion of “resealed” CAMs by antisense clones  grown in culture. 125I-UdR–labeled cells (3 × 105 per CAM) derived from individual clones were inoculated each onto eight  CAMs. Before inoculation the CAMs were “wounded” and then  allowed to reseal for 24 h. The median of invasion is shown for  each group. Comparison of groups by analysis of variance (SYSTAT) analysis showed a highly statistically significant difference  (P = 0.000). A post-hoc analysis of each clone versus LK 25 control showed a value of P = 0.000, except for AS 32, in which P =  0.001. Similar results were obtained with control clones (LK 9  and 12) (not shown).
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Figure 3: Invasion of “resealed” CAMs by antisense clones grown in culture. 125I-UdR–labeled cells (3 × 105 per CAM) derived from individual clones were inoculated each onto eight CAMs. Before inoculation the CAMs were “wounded” and then allowed to reseal for 24 h. The median of invasion is shown for each group. Comparison of groups by analysis of variance (SYSTAT) analysis showed a highly statistically significant difference (P = 0.000). A post-hoc analysis of each clone versus LK 25 control showed a value of P = 0.000, except for AS 32, in which P = 0.001. Similar results were obtained with control clones (LK 9 and 12) (not shown).

Mentions: The invasive ability of the antisense clones was compared to that of control cells by inoculating equal numbers of 125I-UdR–labeled cells onto CAMs that were wounded and allowed to reseal for 24 h before inoculation (Ossowski, 1988). After 24 h of incubation, the number of labeled invading cells was measured and the invasion calculated as percent of total recovered tumor cells (see Materials and Methods). As shown in Fig. 3, while control cell invasion was 68%, the invasion by clones with reduced uPAR level was only 24% or less. This highly statistically significant difference confirms our previous results, which showed that surface uPA is essential for invasion of CAM and of dermis when inoculated subcutaneously in nude mice (Kook et al., 1994).


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

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

Invasion of “resealed” CAMs by antisense clones  grown in culture. 125I-UdR–labeled cells (3 × 105 per CAM) derived from individual clones were inoculated each onto eight  CAMs. Before inoculation the CAMs were “wounded” and then  allowed to reseal for 24 h. The median of invasion is shown for  each group. Comparison of groups by analysis of variance (SYSTAT) analysis showed a highly statistically significant difference  (P = 0.000). A post-hoc analysis of each clone versus LK 25 control showed a value of P = 0.000, except for AS 32, in which P =  0.001. Similar results were obtained with control clones (LK 9  and 12) (not shown).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Invasion of “resealed” CAMs by antisense clones grown in culture. 125I-UdR–labeled cells (3 × 105 per CAM) derived from individual clones were inoculated each onto eight CAMs. Before inoculation the CAMs were “wounded” and then allowed to reseal for 24 h. The median of invasion is shown for each group. Comparison of groups by analysis of variance (SYSTAT) analysis showed a highly statistically significant difference (P = 0.000). A post-hoc analysis of each clone versus LK 25 control showed a value of P = 0.000, except for AS 32, in which P = 0.001. Similar results were obtained with control clones (LK 9 and 12) (not shown).
Mentions: The invasive ability of the antisense clones was compared to that of control cells by inoculating equal numbers of 125I-UdR–labeled cells onto CAMs that were wounded and allowed to reseal for 24 h before inoculation (Ossowski, 1988). After 24 h of incubation, the number of labeled invading cells was measured and the invasion calculated as percent of total recovered tumor cells (see Materials and Methods). As shown in Fig. 3, while control cell invasion was 68%, the invasion by clones with reduced uPAR level was only 24% or less. This highly statistically significant difference confirms our previous results, which showed that surface uPA is essential for invasion of CAM and of dermis when inoculated subcutaneously in nude mice (Kook et al., 1994).

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