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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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Dormancy of antisense-transfected clones maintained in vivo. Cells of individual clones (5 × 105 per  CAM) were inoculated on  CAMs of two 10-d-old chick  embryos and incubated for 1  wk, and the resulting nodules  were excised, weighed, minced,  and reinoculated onto two  fresh CAMs. Serial passage  of tumors were discontinued  when their weight exceeded  100 mg. (We observed that  tumors of this size, which  contain mostly live tumor  cells, usually continue progressive growth). M(−) indicates that lungs of these embryos were tested for  metastases and found to be  negative.
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Figure 4: Dormancy of antisense-transfected clones maintained in vivo. Cells of individual clones (5 × 105 per CAM) were inoculated on CAMs of two 10-d-old chick embryos and incubated for 1 wk, and the resulting nodules were excised, weighed, minced, and reinoculated onto two fresh CAMs. Serial passage of tumors were discontinued when their weight exceeded 100 mg. (We observed that tumors of this size, which contain mostly live tumor cells, usually continue progressive growth). M(−) indicates that lungs of these embryos were tested for metastases and found to be negative.

Mentions: Previous studies of a single uPAR antisense clone suggested that reduced uPAR expression induced tumor cell dormancy (Kook et al., 1994) and that permanent dormancy may be possible to achieve provided uPAR reduction was sustained. To explore this intriguing but preliminary observation, each of the five newly obtained antisense and four control clones was inoculated onto CAMs and maintained by serial weekly passage onto fresh CAMs. After the first week of growth, and weekly thereafter, the CAMs were inspected visually, and the site of inoculation (a clearly visible small nodule arising in response to wounding occurring during preparation of the CAM) was excised. The nodules were weighed, minced, and microscopically inspected for the presence of live tumor cells. (Tumor cells were always discernible in the mince.) In antisense clones, the entire mince, and in the controls part of the mince, was reinoculated onto fresh CAMs. Starting from week 2 on the CAM, each of the control clones produced progressively growing, large tumors (Fig. 4). In contrast, the small nodules produced by the antisense clones either remained completely static or fluctuated slightly, without showing a persistent increase in mass, for as long as 4 to 5 mo (Fig. 4, A–D). One clone (AS 36) initiated growth after 2 mo (not shown). CAMs were inoculated a second time with cells from each of the controls and all but one (AS 33) of the antisense clones and maintained by serial passages for up to 8 wk, yielding essentially the same results as those shown in Fig. 4 for the initial test period of growth on CAMs and indicating that inability to grow in vivo was a reproducible property of the antisense-inhibited cells. Moreover, dormancy was observed already in the first week on CAM, regardless of the number (as low as 1 × 105 or as high as 2 × 106) of inoculated, uPARdeficient cells (results not shown), suggesting that it was the interaction with the host and not tumor cell–cell interaction that was responsible for the effect.


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

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

Dormancy of antisense-transfected clones maintained in vivo. Cells of individual clones (5 × 105 per  CAM) were inoculated on  CAMs of two 10-d-old chick  embryos and incubated for 1  wk, and the resulting nodules  were excised, weighed, minced,  and reinoculated onto two  fresh CAMs. Serial passage  of tumors were discontinued  when their weight exceeded  100 mg. (We observed that  tumors of this size, which  contain mostly live tumor  cells, usually continue progressive growth). M(−) indicates that lungs of these embryos were tested for  metastases and found to be  negative.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Dormancy of antisense-transfected clones maintained in vivo. Cells of individual clones (5 × 105 per CAM) were inoculated on CAMs of two 10-d-old chick embryos and incubated for 1 wk, and the resulting nodules were excised, weighed, minced, and reinoculated onto two fresh CAMs. Serial passage of tumors were discontinued when their weight exceeded 100 mg. (We observed that tumors of this size, which contain mostly live tumor cells, usually continue progressive growth). M(−) indicates that lungs of these embryos were tested for metastases and found to be negative.
Mentions: Previous studies of a single uPAR antisense clone suggested that reduced uPAR expression induced tumor cell dormancy (Kook et al., 1994) and that permanent dormancy may be possible to achieve provided uPAR reduction was sustained. To explore this intriguing but preliminary observation, each of the five newly obtained antisense and four control clones was inoculated onto CAMs and maintained by serial weekly passage onto fresh CAMs. After the first week of growth, and weekly thereafter, the CAMs were inspected visually, and the site of inoculation (a clearly visible small nodule arising in response to wounding occurring during preparation of the CAM) was excised. The nodules were weighed, minced, and microscopically inspected for the presence of live tumor cells. (Tumor cells were always discernible in the mince.) In antisense clones, the entire mince, and in the controls part of the mince, was reinoculated onto fresh CAMs. Starting from week 2 on the CAM, each of the control clones produced progressively growing, large tumors (Fig. 4). In contrast, the small nodules produced by the antisense clones either remained completely static or fluctuated slightly, without showing a persistent increase in mass, for as long as 4 to 5 mo (Fig. 4, A–D). One clone (AS 36) initiated growth after 2 mo (not shown). CAMs were inoculated a second time with cells from each of the controls and all but one (AS 33) of the antisense clones and maintained by serial passages for up to 8 wk, yielding essentially the same results as those shown in Fig. 4 for the initial test period of growth on CAMs and indicating that inability to grow in vivo was a reproducible property of the antisense-inhibited cells. Moreover, dormancy was observed already in the first week on CAM, regardless of the number (as low as 1 × 105 or as high as 2 × 106) of inoculated, uPARdeficient cells (results not shown), suggesting that it was the interaction with the host and not tumor cell–cell interaction that was responsible for the effect.

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