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Site-selective cyclic AMP (cAMP) analogs provide a new approach in the control of breast and colon cancer growth
159s
D-052
A CM'ARATIVE S'IDDY OF ME EFFECTS OF ESTRADIDL-176 ADD Z-OH-ESTRADIOL-17D 011CELLDIVISIaV IW WF-7 AND l&i CELLS Koch, F. and Aveling; N.L. of Physiology, University of Pretoria, Pretoria 0002, South Africa
Seegers, J.C.,vanAswegen, C.H., ~part~nt
The cell proliferating effect of estradiol-178 (EZ) is still poorly understood. Little is also known of the mitotic effects of 2-OHE,, the major metabolite of E,. The role of estrogen receptors (ER) in the initiation of cell division is unclear. We used two cell types; MCF-7 cells (breast cancer cell line) with a high ER population (137 fmol/mg cytosol protein) and WeLa cells (cervix carcinoma cell line) with low ER (43 fmol/mg cytosol protein). The two cell lines reacted in a similar biphasic manner to treatment with Ez. Low levels EZ (< 2,5 x 10-8 M) increased cell growth whereas high dosages of EZ (> 2-5 x 10-6 M) had a pronounced cytotoxic effect on cell division. An average mitotic index (PI.11of 2,2% was observed in haematoxy~in and eosin stained control WCF-7 cells. Treatment with 2,5 x 10-6 M E, caused an increase in M.1 which reached a peak of 9,0% after 16 hours; 5,3% of the 9,0% showed abnormal metaphase configurations. HeLa cell controls had a M.1 of 9.3% and EZ (2,5 x 10-6 M) treatment increased M.1 to 18,7X of which 16,7% were abnormal metaphases (Ab.M) showing uneven chromosome distribution. Indirect innnunofluorescence revealed abnormal spindle formation which explains the uneven chromosome arrangement. MCF-7 and HeLa cells also re= sponded similarly to Z-0HE2 treatment except that a peak in cell division (MCF-7 = 7,3%, Ab.M = 4,5%; HeLa = 20,7%, Ab.M = 9.1%) was reached after only 8 hours. Treatment of 2,5 x 10-7 M Ez exposed cells with 100 UM a-naphthoflavone, an inhibitor of L-hydroxylase, caused a marked reduction of M.1 (3,6%) and abnormal metaphases (Ab.M = 0,6%) in HeLa cells, indicating that Et is possibly metabolized to 2-OWEI before increased cell proliferation takes place, and further= more that high levels of 2-OHEa and not Er are responsible for the abnormal spindle formation. From this study it would also appear that cells with high or low ER populations react similarly to the cytotoxic effects of estradiol.
D-053
REGUT,ATION OF T:IE SYNTi3ESIS OF SECRETED CELL LINE, WCF-7.
PROTEIZS
FROM
THE HUMAi
BREAST
CANCER
Anne E. Lykkesfeldt, Inga Laursen and Per Briand. The Fibiger Institute, Ndr. Frihavnsgade 70, DK-2100 Copenhagen (d, Denmark. The human breast cancer cell line XCF-7 is continuously propagated in DME/F12 medium supplemented with glutamine 2 mu, insulin 6 ng/ml and 0.5% FCS (Subline 0.5) or in DiJIEfFl2medium only supplemented with glutamine 2mX (Subline 9). The generation time for the two sublines are about 24 hours and 48 hours, respectively. Both cell lines are growth inhibited by addition of newborn calf serum (NCS) to their conventional growth medium. The effect of NCS on the secreted proteins is only minor in Subline 9, whereas Subline 0.5 secrets a reduced amount of a 52K protein. Addition of estradiol to the conventional growth media results in only a minor growth stimulation of both cell lines and no detectable change in the pattern of secreted proteins. Estradiol has a pronounced stimulatory effect on cell proliferation when the cell lines are grown with 10% NCS, and the synthesis and secretion of the specific proteins 52X, 65K and 70K are significantly stimulated, whereas the synthesis of a 46K protein is inhibited. The synthesis of the various proteins will be correlated to cell proliferation. D-054
SITE-SRLRCTIVR CYCLICAMP (CAMP)ANALOGSPROVIDRA NEW APPROACH IN TUB CONTROL OF BREAST AND COLON CANCER GROWTH. Cho-Chung, Y.S., Katsaros, D., Ally, S., Tortora, G., Cl&r, T., and Robins, R.K. National Cancer Institute, Bethesda, MD 20892 and Nucleic Acid Research Institute, Costa Mesa, CA 92626. Past studies indicated that the Rxx CAMP receptor protein, the regulatory subunit oE CAMPdependent protein kinase type 11 plays a pivotal role in the control of cancer growth and differentiation. We now show that the site-selective CAMP analogs which are many-fold more active in binding to chMP receptor protein than previously studied analogs, demonstrate a potent growth inhibition OF 8even breast and three colon human cancer cell lines. CAMP functions by binding to the CAMP receptor protein which ha8 two different CAMP binding sites and CAMP analogs that selectively bind to either one of the two sites are known as Site l-selective (C-8 analogs) and Site 2-selective (C-6 analogs), respectively. Nineteen monosubstituted and disubstituted siteselective analogs were tested. The majority of these analogs produced an appreciable gtowth inhibition with no sign of toxicity in all 10 cancer lines at UM concentrations. The three most potent inhibitors were S-Cl-, N6-benxyl-, and N6-phenyl-S-thio-pCl-phenyl-&HP, causing 40-75X inhibition at lo-20 uM concentrations. In a hormone-dependent breast cancer line, 8-Cl-cAMP (1 uM) + B6-benxyl-cAMP (0.5 uM) completely blocked the estrogen stimulation of growth, while tamoxifen (1 uM) produced a 50% inhibition. The growth inhibition did not accompany change in cell cycle phase but paralleled change in cell morphology, augmentation of RII cAUP receptor protein and reduction of ~21 ras protein. The cells treated with the adenosine counterpart of the analogs produced cell killing and Gl block with no effect on the RI1 and p21 ras protein. Slteselective CAMP analogs, thus provide a new biological tool for control of zcer growth. S828:SLmPL-r
D-052
A CM'ARATIVE S'IDDY OF ME EFFECTS OF ESTRADIDL-176 ADD Z-OH-ESTRADIOL-17D 011CELLDIVISIaV IW WF-7 AND l&i CELLS Koch, F. and Aveling; N.L. of Physiology, University of Pretoria, Pretoria 0002, South Africa
Seegers, J.C.,vanAswegen, C.H., ~part~nt
The cell proliferating effect of estradiol-178 (EZ) is still poorly understood. Little is also known of the mitotic effects of 2-OHE,, the major metabolite of E,. The role of estrogen receptors (ER) in the initiation of cell division is unclear. We used two cell types; MCF-7 cells (breast cancer cell line) with a high ER population (137 fmol/mg cytosol protein) and WeLa cells (cervix carcinoma cell line) with low ER (43 fmol/mg cytosol protein). The two cell lines reacted in a similar biphasic manner to treatment with Ez. Low levels EZ (< 2,5 x 10-8 M) increased cell growth whereas high dosages of EZ (> 2-5 x 10-6 M) had a pronounced cytotoxic effect on cell division. An average mitotic index (PI.11of 2,2% was observed in haematoxy~in and eosin stained control WCF-7 cells. Treatment with 2,5 x 10-6 M E, caused an increase in M.1 which reached a peak of 9,0% after 16 hours; 5,3% of the 9,0% showed abnormal metaphase configurations. HeLa cell controls had a M.1 of 9.3% and EZ (2,5 x 10-6 M) treatment increased M.1 to 18,7X of which 16,7% were abnormal metaphases (Ab.M) showing uneven chromosome distribution. Indirect innnunofluorescence revealed abnormal spindle formation which explains the uneven chromosome arrangement. MCF-7 and HeLa cells also re= sponded similarly to Z-0HE2 treatment except that a peak in cell division (MCF-7 = 7,3%, Ab.M = 4,5%; HeLa = 20,7%, Ab.M = 9.1%) was reached after only 8 hours. Treatment of 2,5 x 10-7 M Ez exposed cells with 100 UM a-naphthoflavone, an inhibitor of L-hydroxylase, caused a marked reduction of M.1 (3,6%) and abnormal metaphases (Ab.M = 0,6%) in HeLa cells, indicating that Et is possibly metabolized to 2-OWEI before increased cell proliferation takes place, and further= more that high levels of 2-OHEa and not Er are responsible for the abnormal spindle formation. From this study it would also appear that cells with high or low ER populations react similarly to the cytotoxic effects of estradiol.
D-053
REGUT,ATION OF T:IE SYNTi3ESIS OF SECRETED CELL LINE, WCF-7.
PROTEIZS
FROM
THE HUMAi
BREAST
CANCER
Anne E. Lykkesfeldt, Inga Laursen and Per Briand. The Fibiger Institute, Ndr. Frihavnsgade 70, DK-2100 Copenhagen (d, Denmark. The human breast cancer cell line XCF-7 is continuously propagated in DME/F12 medium supplemented with glutamine 2 mu, insulin 6 ng/ml and 0.5% FCS (Subline 0.5) or in DiJIEfFl2medium only supplemented with glutamine 2mX (Subline 9). The generation time for the two sublines are about 24 hours and 48 hours, respectively. Both cell lines are growth inhibited by addition of newborn calf serum (NCS) to their conventional growth medium. The effect of NCS on the secreted proteins is only minor in Subline 9, whereas Subline 0.5 secrets a reduced amount of a 52K protein. Addition of estradiol to the conventional growth media results in only a minor growth stimulation of both cell lines and no detectable change in the pattern of secreted proteins. Estradiol has a pronounced stimulatory effect on cell proliferation when the cell lines are grown with 10% NCS, and the synthesis and secretion of the specific proteins 52X, 65K and 70K are significantly stimulated, whereas the synthesis of a 46K protein is inhibited. The synthesis of the various proteins will be correlated to cell proliferation. D-054
SITE-SRLRCTIVR CYCLICAMP (CAMP)ANALOGSPROVIDRA NEW APPROACH IN TUB CONTROL OF BREAST AND COLON CANCER GROWTH. Cho-Chung, Y.S., Katsaros, D., Ally, S., Tortora, G., Cl&r, T., and Robins, R.K. National Cancer Institute, Bethesda, MD 20892 and Nucleic Acid Research Institute, Costa Mesa, CA 92626. Past studies indicated that the Rxx CAMP receptor protein, the regulatory subunit oE CAMPdependent protein kinase type 11 plays a pivotal role in the control of cancer growth and differentiation. We now show that the site-selective CAMP analogs which are many-fold more active in binding to chMP receptor protein than previously studied analogs, demonstrate a potent growth inhibition OF 8even breast and three colon human cancer cell lines. CAMP functions by binding to the CAMP receptor protein which ha8 two different CAMP binding sites and CAMP analogs that selectively bind to either one of the two sites are known as Site l-selective (C-8 analogs) and Site 2-selective (C-6 analogs), respectively. Nineteen monosubstituted and disubstituted siteselective analogs were tested. The majority of these analogs produced an appreciable gtowth inhibition with no sign of toxicity in all 10 cancer lines at UM concentrations. The three most potent inhibitors were S-Cl-, N6-benxyl-, and N6-phenyl-S-thio-pCl-phenyl-&HP, causing 40-75X inhibition at lo-20 uM concentrations. In a hormone-dependent breast cancer line, 8-Cl-cAMP (1 uM) + B6-benxyl-cAMP (0.5 uM) completely blocked the estrogen stimulation of growth, while tamoxifen (1 uM) produced a 50% inhibition. The growth inhibition did not accompany change in cell cycle phase but paralleled change in cell morphology, augmentation of RII cAUP receptor protein and reduction of ~21 ras protein. The cells treated with the adenosine counterpart of the analogs produced cell killing and Gl block with no effect on the RI1 and p21 ras protein. Slteselective CAMP analogs, thus provide a new biological tool for control of zcer growth. S828:SLmPL-r