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315 The relationship between a highly metastatic human cell line and plasminogen activator (PA) activity
137
315TAERELATIONSEIP BUMAN CELL (PA) ACTIVITY
LINE
BEYWEEN A BIGELY AND PLASMINOGEN
NETASTATIC ACTIVATOR
*DAPCooke. +Mmi. ++RJ Ormerod. *Department of Surgery, DMDS St Thomas' Hospital, London SE1 7ER. ++1cRF. Lincolns +NIBSC South Himms. Berts. Inn Fields, London WC2 Plasminogen activators, particularly urokinase the spread of (uPA), have been implicated tumours. The relationship between PA activity and metastatic spread in 3 human cell lines has been examined in vitro and in vivo. DX3-LT5.1 is derived from a human melanoma
cell line, DX3, by treatment with the 5-azacytidine. a known nucleoside analogue, activator of gene expression. A431 is a well Three groups of known epidermal cancer line. athymic mice were injected i.v. with 5 x 105 No lung cells and sacrificed six weeks later. metastases were visible in the A431 or DX3 groups but in the group injected with the
LT5.1
sub-line
seen.(Mean
33;
107 cells were added to seven ceil lines, flasks in 10% FCS-conditioned medium. 80cm2 After 12 hours the monolayers were gently for washed and charged with serum free medium were a further 24 hours when the cells The cell pellets were harvested and counted. into PBS and extracted cryofragmented, A bioimmunoassay (BIA) was used centrifuged. to measure the PA activity of the supernatent. The A431 line contained little tPA but large cells; amounts of uPA (tPA 0.59 2 0.13 iu/lOa UPA 8.42 + 1.53 iu/lOe cells) whereas the DX3, contained higher levels of tPA and lower of uPA (tPA 20.18 + 4.87; UPA 0.13 + levels DX3-LT5.1, 0.08) The highly metastatic line, uPA contained less activity (tPA 6.31 + 2.57; line, 0.05 + 0.03) than the low metastatic The increased uPA DX3, from which it derived. levels associated with human breast and colon metastatic do not correlate with cancer potential in this animal model.
a considerable number was range l-76). Of each of the 3
316INCREASED PRODUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE-l (PAI-1) IN HUMAN NEOPLASTIC CELL LINES EXPOSED TO TUMORPROMOTIN~,EJIORBOL E;ST;H. M. Mayer , . . Riccio3. K. I. A. L
Dandl and P.A. Andreasen ;Finsen Laboratory, Institute of Biochemistry tute of Microbiology, Copenhagen.
'
. Rigshospjtalet; C and InstiUniversity of
Using a sensitive and specific ELISA, we measured the effect of phorbol 12myristate 13-acetate (PMA) on PAIlevels in 15 human neoplastic cell lines. Cell lines which undergo a PMA-dependent differentiation (HL-60, U937) responded to PMA with a marked accumulation of PAIprotein in the culture medium. Also, some lines which do not differenreponse to PMA nevertheless tiate in showed a PMA-induced increase in secreted
3~7IMMUNOCYTOCHEMICAL DEMONSTRATION OF t-PA IN BENIGN AND MALIGNANT BREAST . . TISSUE. +Sadra *D A P S&&s. +Ruth -es. . Larkin +AMRmes. *K G B-and. **M w. +Eam Beydw
.Departments of *Surgery and 'olog~. UMDS, St Thomas' Bospital, VEH, **NIHSC South Mimms, Herts. Plasminogen activators the in spread of examine the expression
+HistopathLondon SE1
have been implicated malignant tumours. To of t-PA in benign and malignant breast tissues we raised rabbit antibodies using pure polyclonal human t-PA (Ciba-Geigy, melanoma Basle) and studied 48 breast lesions: 6 were fibroadenomas and 14 fibrocystic (4 of these were near carcinoma). 28 carcinomas were studied. Methacarn-fixed, paraffinembedded sections of the tissues were stained using :an indirect -immunoperoxidase technique. :Adjacent tissues were snapfrozen and cryofragmented. The t-PA was extracted into PBS and functional t-PA activity was measured by a bioimmunoassay (BIA). All of
PAIprotein level. Investigating the mechanism of this induction in selected cell lines, we found that the effect was half-maximal at lo-20 nM PMA, and was specific for tumor-promoting pharbols known to activate protein kinase C. The increase in medium level of PAIwas preceded by a transient increase in intracellular PAI-1, maximal at 16 hours after addition of PMA. Northernand dotblot hybridization with PAIcDNA showed that PMA produces a 20-fold increase in the steady-state level of the specific mRNA. The relative abundance of a 2.2 Kb and a 3.1 Eb transcript changes with time following PMA. We conclude that tumor promoters increase expression of the PAIgene.
the. benign and .18/28 of the malignant samples stained .positively although the benign tissue staining pattern was more heterogeneous. Blood vessel endothelium acted as a positive control. The carcinomas showed activity of 45.97iu/gm (SD+67.0;~an~~a~-268) and the benign tissues a mean activity of 35,76iu/gm (SD+Bl.B;Range 4.9-84) Whilst the BIA showed no significant difference (p= >0.5) between the groups, the malignant samples were more cellular and the staining in 12/18 of them was weak, whereas the benign showed a more heterogeneous pattern of staining. Such staining will show individual cellular activity more accurately than assay of the homogenate and may reflect differences in function, differentiation or cell cycle. Information from both techniques may be important in examining the relationship fibrinolysis and between malignancy.
315TAERELATIONSEIP BUMAN CELL (PA) ACTIVITY
LINE
BEYWEEN A BIGELY AND PLASMINOGEN
NETASTATIC ACTIVATOR
*DAPCooke. +Mmi. ++RJ Ormerod. *Department of Surgery, DMDS St Thomas' Hospital, London SE1 7ER. ++1cRF. Lincolns +NIBSC South Himms. Berts. Inn Fields, London WC2 Plasminogen activators, particularly urokinase the spread of (uPA), have been implicated tumours. The relationship between PA activity and metastatic spread in 3 human cell lines has been examined in vitro and in vivo. DX3-LT5.1 is derived from a human melanoma
cell line, DX3, by treatment with the 5-azacytidine. a known nucleoside analogue, activator of gene expression. A431 is a well Three groups of known epidermal cancer line. athymic mice were injected i.v. with 5 x 105 No lung cells and sacrificed six weeks later. metastases were visible in the A431 or DX3 groups but in the group injected with the
LT5.1
sub-line
seen.(Mean
33;
107 cells were added to seven ceil lines, flasks in 10% FCS-conditioned medium. 80cm2 After 12 hours the monolayers were gently for washed and charged with serum free medium were a further 24 hours when the cells The cell pellets were harvested and counted. into PBS and extracted cryofragmented, A bioimmunoassay (BIA) was used centrifuged. to measure the PA activity of the supernatent. The A431 line contained little tPA but large cells; amounts of uPA (tPA 0.59 2 0.13 iu/lOa UPA 8.42 + 1.53 iu/lOe cells) whereas the DX3, contained higher levels of tPA and lower of uPA (tPA 20.18 + 4.87; UPA 0.13 + levels DX3-LT5.1, 0.08) The highly metastatic line, uPA contained less activity (tPA 6.31 + 2.57; line, 0.05 + 0.03) than the low metastatic The increased uPA DX3, from which it derived. levels associated with human breast and colon metastatic do not correlate with cancer potential in this animal model.
a considerable number was range l-76). Of each of the 3
316INCREASED PRODUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR TYPE-l (PAI-1) IN HUMAN NEOPLASTIC CELL LINES EXPOSED TO TUMORPROMOTIN~,EJIORBOL E;ST;H. M. Mayer , . . Riccio3. K. I. A. L
Dandl and P.A. Andreasen ;Finsen Laboratory, Institute of Biochemistry tute of Microbiology, Copenhagen.
'
. Rigshospjtalet; C and InstiUniversity of
Using a sensitive and specific ELISA, we measured the effect of phorbol 12myristate 13-acetate (PMA) on PAIlevels in 15 human neoplastic cell lines. Cell lines which undergo a PMA-dependent differentiation (HL-60, U937) responded to PMA with a marked accumulation of PAIprotein in the culture medium. Also, some lines which do not differenreponse to PMA nevertheless tiate in showed a PMA-induced increase in secreted
3~7IMMUNOCYTOCHEMICAL DEMONSTRATION OF t-PA IN BENIGN AND MALIGNANT BREAST . . TISSUE. +Sadra *D A P S&&s. +Ruth -es. . Larkin +AMRmes. *K G B-and. **M w. +Eam Beydw
.Departments of *Surgery and 'olog~. UMDS, St Thomas' Bospital, VEH, **NIHSC South Mimms, Herts. Plasminogen activators the in spread of examine the expression
+HistopathLondon SE1
have been implicated malignant tumours. To of t-PA in benign and malignant breast tissues we raised rabbit antibodies using pure polyclonal human t-PA (Ciba-Geigy, melanoma Basle) and studied 48 breast lesions: 6 were fibroadenomas and 14 fibrocystic (4 of these were near carcinoma). 28 carcinomas were studied. Methacarn-fixed, paraffinembedded sections of the tissues were stained using :an indirect -immunoperoxidase technique. :Adjacent tissues were snapfrozen and cryofragmented. The t-PA was extracted into PBS and functional t-PA activity was measured by a bioimmunoassay (BIA). All of
PAIprotein level. Investigating the mechanism of this induction in selected cell lines, we found that the effect was half-maximal at lo-20 nM PMA, and was specific for tumor-promoting pharbols known to activate protein kinase C. The increase in medium level of PAIwas preceded by a transient increase in intracellular PAI-1, maximal at 16 hours after addition of PMA. Northernand dotblot hybridization with PAIcDNA showed that PMA produces a 20-fold increase in the steady-state level of the specific mRNA. The relative abundance of a 2.2 Kb and a 3.1 Eb transcript changes with time following PMA. We conclude that tumor promoters increase expression of the PAIgene.
the. benign and .18/28 of the malignant samples stained .positively although the benign tissue staining pattern was more heterogeneous. Blood vessel endothelium acted as a positive control. The carcinomas showed activity of 45.97iu/gm (SD+67.0;~an~~a~-268) and the benign tissues a mean activity of 35,76iu/gm (SD+Bl.B;Range 4.9-84) Whilst the BIA showed no significant difference (p= >0.5) between the groups, the malignant samples were more cellular and the staining in 12/18 of them was weak, whereas the benign showed a more heterogeneous pattern of staining. Such staining will show individual cellular activity more accurately than assay of the homogenate and may reflect differences in function, differentiation or cell cycle. Information from both techniques may be important in examining the relationship fibrinolysis and between malignancy.