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Plasminogen Activator Production by Human Prostate Carcinoma Cell Lines
Accepted 42
41 ELEVATE~ TRANSFERRIN REC~PTORS(TfR) IN PROSTATIC CANCER(PCa) CELLS.
Harold N. Keer,
Yvonne Tsai, *James M. Kozlowski,
and John T. Grayhack, Chicago, IL. (Presentation to be made by Mr. Keer) Elevated levels of transferrin(Tf) have been noted in the prostatic fluid of PCa patients. Hypothesizing that this increase·may be the result of receptor mediated sequestration, we measured TfR content in a variety of
benign and malignant prostatic cells. We studied: 1) established PCa cell lines, 2)benign prostatic hyperplasia(BPH) stromal cells grown in tissue culture, 3)fresh BPH tissue, and !•)fresh nude mouse tumors resulting from the injection
of established PCa cells. The fresh tissue was enzymatically dissociated to a single Cell suspensiOn and the stromal and epithelial components separated using a discontinuous Percell gradient. Human placenta from term pregnancy and human erythrocytes were used as posltiVe and negative con-
trols, respectively. Scatchard analysis using 125 1-Tf was performed on Triton X-100 solubilized membrane extracts to
assess TfR content. Two established PCa cell lines, PC3 (bone marrow metastasis derived) and DU145( brain metastasis derived) showed significant TfR content (PC3-12 fmoles/ug protein, DU145-7. 5 fmoles/ug) while the cultured BPH cells(stromal) had no binding above that of the-negative control. The strOmal elements of fresh BPH and tumor tissues had no detectable TfR above background while the BPH epithelium had low (0.3-0.7 fmole/ug) but measurable amounts of TfR. Epithelial cells derived from the tumors grown in.nude mice had TfR levels similar to those of the established PCa cell lines in tissue culture. These levels were 14 times higher than those of the BPH epitheliUm. These results provide evidence that the TfR content is iUcreased in PCa comPared to BPH and is restricted to the epithelial cells. Further, the data support the concept that the elevated Tf in prostatic fluid in PCa patients is linked to an increase in TfR in the cancerous cells,
BIOLCGICAL SIGNIFICANCE OF PROSTATIC CARCINOMA AFTER DEFINATIVE RADIATION THERAPY. *Paul w. Musselman' *Rayrrnnd Tubbs' Robert W. Connell yj Takayuki Hashimura1 J. &!son Pontes. Cleveland, OH (Presentation to be made by Dr. Musselman) Although controversies have existed regarding the viability and biological potential of prostatic cancer cells after definative radiation therapy evidence l:cth clinical and laboratorial is accumulating suggesting that these cells are viable and capable of rretastatses. This study was initiated as an attempt to define in vitro characteristics which prove the viability of these cells. ':lwelve patients with localized prostatic cancer (6 previously radiated rrore than two years previously, and 6 nonradiated) underwent radical prostatectany as =ative treabrent. All specirnens were rerroved under sterile conditions from areas known to contain cancer. Lal::oratory parameters evaluated to confinn viability and biological characterization of prostate indulded: rronolayer growth, in vitro imnunohistochemical identificaiton of cells growing in culture by antitodies to PAP, PSA, and Cytokeratines growth in soft agar, FCM and karyotype. Results ~specimens radiated and non-radiated exhibited nonolayer growth from explants, with rrorphological characteristics of epithelial cells. All cells growing .in culture.derronstrated specific immunochemical staining either to PAP, PSA, or l:cth. Imnunohistochemistry characterization of cytokeratine was positive in one of the tested radiated specimens. Soft: agar colony fonnation was present in one specimen tested. DNA flow cytometry in two radiated specimens derronstrated a diploid pattern. In one of these specimens karyotype analysis (A. Sandberg, Buffalo New York) showed a hypodiploid m::xle, with several chrorrosarnal abnonnalities. The findings in this study are strong evidence that the presence of turror cells tw::i years after definitive radiation therapy continue to exhibit characteristics of viability.
43
44
P1ASMINOGEN ACTIVATOR PRODUCTION BY HUMAN PROSTATE CARCINOMA CELL LINES. Franklin D. Gaylis; Chicago IL; Robert McKewan;; Kalamazoo MI; Al
UROKINASE: A SPECIFIC MARKJ!l. OF MFJ'ASTASATION IN PRO~TATIC CARCINOMA. Hetnz. Pflliger, Johannes c. Kirchheimer, Greff,Or Hienert, Bernd R. Binder, Vienna, Austria lPresentation to be made by Dr. Pflliger) Urokinase-type plasminogen activator ( u-PA), a serine protease able to convert the zymogen plasminogen into plasmin, is elevated in twnor tissue of carcinomas of the urogenital tract as compared to their respective normal counterparts. The same enzyme can also be found at very low concentrations in plasma by means of a radioimmunoassay. However; no correlation between u-PA present in the prostatic carcinoma tissue and in plasma could be found. The appearance of high amounts of u-PA in tumors has been implicated with .the processes of twnor invasion and metastasation, accompanied by the finding of markedly increased u-PA levels in bone metastases of prostatic carcinomas. Based on these findings we determined the enzyme in plasma of patients with prostatic carcinomas with (PCM, n=13) and without metastases (PC, n=23) and compared the results with acid phosphatase, prostate specific acid phosphatase and alkaline phosphatase, three well known tumor markers, routinely used in the detection and follow up of prostatic carcinoma. PC PCM * u-PA 0.86 + 0.25 1 .09 :t 0.25* alkaline phosphatase 87.00 + 19.99 263 .09 :t 224. 93* acid phosphatase 4.92 + 1 .92 19.57 :t 22.07* prost.spec.acid phos. 6.67 + 6.05 0.85 0.77 Values represent means± SD; * p<0.05. -
studies were:
Cell extract activity Conditioned media activity (U/107 cells) (U/ml) 88/30 PC3/DUllf5 76/37 Electrophoretic zym::,gr,anis of cultur>e media and cell extracts showed two prominent activator activities of 47 and 51 kilodaltons(KD).In addition PC3 cell extracts had a third narrow band of activator at 42KD.Thus,these tWIDr cells appear to produce predominantly lower molecular, weight activator of the urokinase type.The rrore aggressive PC3 cell line contained and secreted more PA activator than the relatively indolent DU145 line.These findings provide further evidence that PA production ITBY be related to the metastatic capacity of aggressive tumor cells ,and may be an important factor in the diverse biological behavior of prostate cancer. Cell line
+
Similar to the three established tumor markers, a significant increase of plasma u-PA was found in case of metastasing carcinomas as compared to prostatic carcinomas without metastases. Therefore, u-PA seems to be a helpful additional marker for the follow up of the progression in prostatic carcinomas.
114A
41 ELEVATE~ TRANSFERRIN REC~PTORS(TfR) IN PROSTATIC CANCER(PCa) CELLS.
Harold N. Keer,
Yvonne Tsai, *James M. Kozlowski,
and John T. Grayhack, Chicago, IL. (Presentation to be made by Mr. Keer) Elevated levels of transferrin(Tf) have been noted in the prostatic fluid of PCa patients. Hypothesizing that this increase·may be the result of receptor mediated sequestration, we measured TfR content in a variety of
benign and malignant prostatic cells. We studied: 1) established PCa cell lines, 2)benign prostatic hyperplasia(BPH) stromal cells grown in tissue culture, 3)fresh BPH tissue, and !•)fresh nude mouse tumors resulting from the injection
of established PCa cells. The fresh tissue was enzymatically dissociated to a single Cell suspensiOn and the stromal and epithelial components separated using a discontinuous Percell gradient. Human placenta from term pregnancy and human erythrocytes were used as posltiVe and negative con-
trols, respectively. Scatchard analysis using 125 1-Tf was performed on Triton X-100 solubilized membrane extracts to
assess TfR content. Two established PCa cell lines, PC3 (bone marrow metastasis derived) and DU145( brain metastasis derived) showed significant TfR content (PC3-12 fmoles/ug protein, DU145-7. 5 fmoles/ug) while the cultured BPH cells(stromal) had no binding above that of the-negative control. The strOmal elements of fresh BPH and tumor tissues had no detectable TfR above background while the BPH epithelium had low (0.3-0.7 fmole/ug) but measurable amounts of TfR. Epithelial cells derived from the tumors grown in.nude mice had TfR levels similar to those of the established PCa cell lines in tissue culture. These levels were 14 times higher than those of the BPH epitheliUm. These results provide evidence that the TfR content is iUcreased in PCa comPared to BPH and is restricted to the epithelial cells. Further, the data support the concept that the elevated Tf in prostatic fluid in PCa patients is linked to an increase in TfR in the cancerous cells,
BIOLCGICAL SIGNIFICANCE OF PROSTATIC CARCINOMA AFTER DEFINATIVE RADIATION THERAPY. *Paul w. Musselman' *Rayrrnnd Tubbs' Robert W. Connell yj Takayuki Hashimura1 J. &!son Pontes. Cleveland, OH (Presentation to be made by Dr. Musselman) Although controversies have existed regarding the viability and biological potential of prostatic cancer cells after definative radiation therapy evidence l:cth clinical and laboratorial is accumulating suggesting that these cells are viable and capable of rretastatses. This study was initiated as an attempt to define in vitro characteristics which prove the viability of these cells. ':lwelve patients with localized prostatic cancer (6 previously radiated rrore than two years previously, and 6 nonradiated) underwent radical prostatectany as =ative treabrent. All specirnens were rerroved under sterile conditions from areas known to contain cancer. Lal::oratory parameters evaluated to confinn viability and biological characterization of prostate indulded: rronolayer growth, in vitro imnunohistochemical identificaiton of cells growing in culture by antitodies to PAP, PSA, and Cytokeratines growth in soft agar, FCM and karyotype. Results ~specimens radiated and non-radiated exhibited nonolayer growth from explants, with rrorphological characteristics of epithelial cells. All cells growing .in culture.derronstrated specific immunochemical staining either to PAP, PSA, or l:cth. Imnunohistochemistry characterization of cytokeratine was positive in one of the tested radiated specimens. Soft: agar colony fonnation was present in one specimen tested. DNA flow cytometry in two radiated specimens derronstrated a diploid pattern. In one of these specimens karyotype analysis (A. Sandberg, Buffalo New York) showed a hypodiploid m::xle, with several chrorrosarnal abnonnalities. The findings in this study are strong evidence that the presence of turror cells tw::i years after definitive radiation therapy continue to exhibit characteristics of viability.
43
44
P1ASMINOGEN ACTIVATOR PRODUCTION BY HUMAN PROSTATE CARCINOMA CELL LINES. Franklin D. Gaylis; Chicago IL; Robert McKewan;; Kalamazoo MI; Al
UROKINASE: A SPECIFIC MARKJ!l. OF MFJ'ASTASATION IN PRO~TATIC CARCINOMA. Hetnz. Pflliger, Johannes c. Kirchheimer, Greff,Or Hienert, Bernd R. Binder, Vienna, Austria lPresentation to be made by Dr. Pflliger) Urokinase-type plasminogen activator ( u-PA), a serine protease able to convert the zymogen plasminogen into plasmin, is elevated in twnor tissue of carcinomas of the urogenital tract as compared to their respective normal counterparts. The same enzyme can also be found at very low concentrations in plasma by means of a radioimmunoassay. However; no correlation between u-PA present in the prostatic carcinoma tissue and in plasma could be found. The appearance of high amounts of u-PA in tumors has been implicated with .the processes of twnor invasion and metastasation, accompanied by the finding of markedly increased u-PA levels in bone metastases of prostatic carcinomas. Based on these findings we determined the enzyme in plasma of patients with prostatic carcinomas with (PCM, n=13) and without metastases (PC, n=23) and compared the results with acid phosphatase, prostate specific acid phosphatase and alkaline phosphatase, three well known tumor markers, routinely used in the detection and follow up of prostatic carcinoma. PC PCM * u-PA 0.86 + 0.25 1 .09 :t 0.25* alkaline phosphatase 87.00 + 19.99 263 .09 :t 224. 93* acid phosphatase 4.92 + 1 .92 19.57 :t 22.07* prost.spec.acid phos. 6.67 + 6.05 0.85 0.77 Values represent means± SD; * p<0.05. -
studies were:
Cell extract activity Conditioned media activity (U/107 cells) (U/ml) 88/30 PC3/DUllf5 76/37 Electrophoretic zym::,gr,anis of cultur>e media and cell extracts showed two prominent activator activities of 47 and 51 kilodaltons(KD).In addition PC3 cell extracts had a third narrow band of activator at 42KD.Thus,these tWIDr cells appear to produce predominantly lower molecular, weight activator of the urokinase type.The rrore aggressive PC3 cell line contained and secreted more PA activator than the relatively indolent DU145 line.These findings provide further evidence that PA production ITBY be related to the metastatic capacity of aggressive tumor cells ,and may be an important factor in the diverse biological behavior of prostate cancer. Cell line
+
Similar to the three established tumor markers, a significant increase of plasma u-PA was found in case of metastasing carcinomas as compared to prostatic carcinomas without metastases. Therefore, u-PA seems to be a helpful additional marker for the follow up of the progression in prostatic carcinomas.
114A