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Infectivity and Expression of Viral Mediated Transfer of Specific Genes Into Prostatic Cancer and Benign Prostatic Hyperplastic Cells
Accepted 45
46
EXTRA-HORMONAL EFFECTS OF IMIDAZOLES ON HUMAN PROSTATIC CANCER. John Trachtenberg and Tobias Eichenberger*, Toronto, Ontario. (Presentation to be made by Dr. Trachtenberg) Ketoconazole(K) is a cytochrome P450 inhibitor that has been demonstrated effective in reducing serum
TREATMENT OF THE DUNNING R3327-G PROSTATIC ADENOCARCINOMA BY HYPERTHERMIA. Norman L. Block, Charles F. Gottlieb*, Miami, FL, G. Burton Seibert*, Dallas, TX (Presentation to be made by Dr. Block) Single dose hyperthermia was delivered to animals implanted with the Dunning R3327-G tumor at one of five temperatures for several times (15, 30 or 60 minutes) (15 groups). Tumors were randomly assigned to either treatment or control. Tumor growth was determined by three-axis caliper measurements. Tumor volumes over time were fit by least squares regre~sion to a quadratic growth model; log (v) =a+ b*t + c*t ,wherev is the tumor volume at time t after treatment. This equation was solved for the time to first doubling (i.e., the time when the volume reached twice that at the time of treatment). The time to first doubling when analyzed by hyperthermia dose expressed as time and temperature growth median values {product limit survival estimates) ranging from 3 days (controls) to a maximum of about 25 days. For each treatment time, increasing the temperature produced a corresponding increase in the median time to first doubling; 15 minutes produced 5.8, 7.7, 9.1, 11.4, and 18.1 days; 30 minutes resulted in 7.8, 7.9, 14.8, 13.2 and 23.6 days; and 60 minutes gave 7.4, 17.8, 25.1, 21.0, and 24.9 days; for 42° to 46°, respectively. Alternatively, the time to first doubling was analyzed by hyperthermia dose expressed as ET43 (equivalent minutes at 43°C). The median times to first doubling (product limit survival estimates) range from O - 22 days, with an apparent plateau (at 22 days) being reached after about 250 minutes ET43. The rate of tumor growth at the time of first doubling was found to be independent ofhyperthermia dose. Hyperthermia treatment (single dose) in this system produces a dose-dependent delay in tumor growth with an apparent limit at 22-25 days (median time to first doub1 ing).
androgens.
Patients with
advanced
prostatic
cancer
treated with K experience the palliative benefits usually seen with other forms of androgen ablation. Several reports have now described that approximately 50% of patients who fail conventional androgen ablation obtain a secondary remission when treated with ketoconazole.
In
order to determine if K might have an extra-hormonal mode of action on prostatic cancer cells, human urogenital
tumors (non androgen dependent prostate PC-3, DU 145; bladder HTB 1,2,4, 5, 9; renal HTB 44) and normal skin fibroblasts were incubated with K (0-100 ug/ml) in both conventional monolayer and methylcellulose based clonogenic systems. Cells or colonies were counted
at
day 1, 2, 3, 5, and 8. At clinically tolerable levels of K (1.0 - 10.0 ug/ml) K significantly reduced prostate cancer (PC-3 and DU-145) viability at all days (e.g. day 8 count: 1.0, 2.5, and 5.0 ug/ml = 33, 18, and 11% viability respectively). There were insignificant effects on the other tumor systems and skin fibroblasts. K appears to have a direct and specific cytotoxic effect on prostatic cancer apart from any hormonal effects. It may
have a role
as a part of combination therapy for
advanced disease.
47
48
INFECTIVITY ANU EXPRESSIUN OF VIRAL MEDIATED TRANSFER OF SPECIFIC GENES INTO PROSTATIC CANCER AND BENIGN PROSTATIC HYPERPLASTIC CELLS. *Paul H. Lee, *Christie Holland, *Joel Greenberger and Mani Menon, Worcester, MA (Presentation to be made by Dr. Lee) We previously have shown that transfer of the gene encoding neomycin resistance (neo) by a retroviral vector (psvx-neo ) can confer drug resistance to G418 (Geneticin) in both prostatic cancer cell lines (Pc-3, DU-145) and BPH cells. We now report on the infectivity and expression of this introduced gene, using psvx packaged into amphotropic murine leukemia viral envelope and produced from NIH-3T3 mouse fibroblasts. Drug resistant PC-3-psvx, DU-145-psvx and BPH-psvx colonies were isolated and maintained in tissue cultures. Southern blotting analysis demonstrated that on an average each cell line contained a single copy of the retroviral vector. The cells were resistant to high levels of G418, demonstrating high levels of expression of neo gene. The supernatant from none of the cell lines was able to produce infectious viral particles when tested on Hela cells. These studies have shown that a retroviral vector can be introduced into prostatic cancer cells and BPH cells, be integrated into the host cell genome and be expressed with high levels of RNA transcribed from the gene. Cellular transformations induced by retroviral infection have been shown to be dependent upon the type of viral vec--to.i,.s used for infection (Feuerman, Un.of Cal., Irvint:). Using the described model, we have shown that the Frpsvx virus is a stronger infectious agent in prostatic cancer cells. We are currently searching for stronger enhancer viruses, e.g. adenovirus and cytomegalovirus to increase infectivity and expression. In addition, we are testing the effect of transferring and expressing an adenovirus with ElA gene which has a function in immortalizing rodent cells (Hou11eling, Virolo~y, lOLl:537,1980). By introducing the ElA gene into fresh prost3tic tissue, we hope to determine whether BPH ce 11 s can be i:;1,.10rta 1ized.
KINETIC EVALUATION OF IMMUNOREGULATORY MARKERS EFFECTED BY TUMOR DEVELOPMENT AND CYTOTOXIC THERAPY. Marvin Rubenstein* and Alvin Dubin*, Chicago, Illinois (Presentation to be made by Dr. Rubenstein). Immune functions are adversely effected by both tumor development and cytotoxic chemotherapy. Tumor influenced immune alterations have been demonstrated in humans bearing prostatic cancers and in animals with the Dunning R3327 prostatic adenocarcinoma. We report a sequential analysis
which kinetically demonstrates the effects of tumor development and chemotherapy upon immune markers. Such analysis identifies effective doses and cycles of cytotoxic drugs effective against tumors, yet having effects upon the im-
mune system.
Cyclophosphamide (CTX) was chosen because at
low doses it synergistically interacts with the immune sy-
stem. Rats bearing or not bearing the R3327 MAT-LyLu variant of the Dunning tumor were evaluated for immune markers
following tumor implantation and CTX therapy. Splenic leukocytes were assayed for T cell subsets with monoclonal antibodies and adherent cell production of E series prostaglandins (PGE) by thin layer chromatography. RESULTS Time elapsed %Helper T %Suppressor T H/S %Total T %Monocytes Day O (Normal) 54.7 46.0 1.20 76.7 22,9 Day 5 37.6 33.8 1.11 72.4 19.4 Day 19-1 X CTX 35.0 33.8 1.04 68.8 20.0 Day 26-2 X CTX 36.3 31.3 1.17 67.D 19.0 Tumor produced alterations of T cell subsets are complete
5 days following implantation. A single CTX dose (3Dmg/Kg), further suppressed the helper/suppressor (H/S) T cell ratio, but a second dose, one week later restored a normal H/S
ratio, despite reductions CTX produced in each subset. CTX produced dose dependent reductions in Total, Helper and Suppressor T cell subsets in both normal and tumor bearing animals. Monocytic representation is uneffected by CTX and PGE production (immunosuppressive) by adherent spleen cells was not lowered. Presumably CTX synergy with the immune system acts at different levels.
115A
46
EXTRA-HORMONAL EFFECTS OF IMIDAZOLES ON HUMAN PROSTATIC CANCER. John Trachtenberg and Tobias Eichenberger*, Toronto, Ontario. (Presentation to be made by Dr. Trachtenberg) Ketoconazole(K) is a cytochrome P450 inhibitor that has been demonstrated effective in reducing serum
TREATMENT OF THE DUNNING R3327-G PROSTATIC ADENOCARCINOMA BY HYPERTHERMIA. Norman L. Block, Charles F. Gottlieb*, Miami, FL, G. Burton Seibert*, Dallas, TX (Presentation to be made by Dr. Block) Single dose hyperthermia was delivered to animals implanted with the Dunning R3327-G tumor at one of five temperatures for several times (15, 30 or 60 minutes) (15 groups). Tumors were randomly assigned to either treatment or control. Tumor growth was determined by three-axis caliper measurements. Tumor volumes over time were fit by least squares regre~sion to a quadratic growth model; log (v) =a+ b*t + c*t ,wherev is the tumor volume at time t after treatment. This equation was solved for the time to first doubling (i.e., the time when the volume reached twice that at the time of treatment). The time to first doubling when analyzed by hyperthermia dose expressed as time and temperature growth median values {product limit survival estimates) ranging from 3 days (controls) to a maximum of about 25 days. For each treatment time, increasing the temperature produced a corresponding increase in the median time to first doubling; 15 minutes produced 5.8, 7.7, 9.1, 11.4, and 18.1 days; 30 minutes resulted in 7.8, 7.9, 14.8, 13.2 and 23.6 days; and 60 minutes gave 7.4, 17.8, 25.1, 21.0, and 24.9 days; for 42° to 46°, respectively. Alternatively, the time to first doubling was analyzed by hyperthermia dose expressed as ET43 (equivalent minutes at 43°C). The median times to first doubling (product limit survival estimates) range from O - 22 days, with an apparent plateau (at 22 days) being reached after about 250 minutes ET43. The rate of tumor growth at the time of first doubling was found to be independent ofhyperthermia dose. Hyperthermia treatment (single dose) in this system produces a dose-dependent delay in tumor growth with an apparent limit at 22-25 days (median time to first doub1 ing).
androgens.
Patients with
advanced
prostatic
cancer
treated with K experience the palliative benefits usually seen with other forms of androgen ablation. Several reports have now described that approximately 50% of patients who fail conventional androgen ablation obtain a secondary remission when treated with ketoconazole.
In
order to determine if K might have an extra-hormonal mode of action on prostatic cancer cells, human urogenital
tumors (non androgen dependent prostate PC-3, DU 145; bladder HTB 1,2,4, 5, 9; renal HTB 44) and normal skin fibroblasts were incubated with K (0-100 ug/ml) in both conventional monolayer and methylcellulose based clonogenic systems. Cells or colonies were counted
at
day 1, 2, 3, 5, and 8. At clinically tolerable levels of K (1.0 - 10.0 ug/ml) K significantly reduced prostate cancer (PC-3 and DU-145) viability at all days (e.g. day 8 count: 1.0, 2.5, and 5.0 ug/ml = 33, 18, and 11% viability respectively). There were insignificant effects on the other tumor systems and skin fibroblasts. K appears to have a direct and specific cytotoxic effect on prostatic cancer apart from any hormonal effects. It may
have a role
as a part of combination therapy for
advanced disease.
47
48
INFECTIVITY ANU EXPRESSIUN OF VIRAL MEDIATED TRANSFER OF SPECIFIC GENES INTO PROSTATIC CANCER AND BENIGN PROSTATIC HYPERPLASTIC CELLS. *Paul H. Lee, *Christie Holland, *Joel Greenberger and Mani Menon, Worcester, MA (Presentation to be made by Dr. Lee) We previously have shown that transfer of the gene encoding neomycin resistance (neo) by a retroviral vector (psvx-neo ) can confer drug resistance to G418 (Geneticin) in both prostatic cancer cell lines (Pc-3, DU-145) and BPH cells. We now report on the infectivity and expression of this introduced gene, using psvx packaged into amphotropic murine leukemia viral envelope and produced from NIH-3T3 mouse fibroblasts. Drug resistant PC-3-psvx, DU-145-psvx and BPH-psvx colonies were isolated and maintained in tissue cultures. Southern blotting analysis demonstrated that on an average each cell line contained a single copy of the retroviral vector. The cells were resistant to high levels of G418, demonstrating high levels of expression of neo gene. The supernatant from none of the cell lines was able to produce infectious viral particles when tested on Hela cells. These studies have shown that a retroviral vector can be introduced into prostatic cancer cells and BPH cells, be integrated into the host cell genome and be expressed with high levels of RNA transcribed from the gene. Cellular transformations induced by retroviral infection have been shown to be dependent upon the type of viral vec--to.i,.s used for infection (Feuerman, Un.of Cal., Irvint:). Using the described model, we have shown that the Frpsvx virus is a stronger infectious agent in prostatic cancer cells. We are currently searching for stronger enhancer viruses, e.g. adenovirus and cytomegalovirus to increase infectivity and expression. In addition, we are testing the effect of transferring and expressing an adenovirus with ElA gene which has a function in immortalizing rodent cells (Hou11eling, Virolo~y, lOLl:537,1980). By introducing the ElA gene into fresh prost3tic tissue, we hope to determine whether BPH ce 11 s can be i:;1,.10rta 1ized.
KINETIC EVALUATION OF IMMUNOREGULATORY MARKERS EFFECTED BY TUMOR DEVELOPMENT AND CYTOTOXIC THERAPY. Marvin Rubenstein* and Alvin Dubin*, Chicago, Illinois (Presentation to be made by Dr. Rubenstein). Immune functions are adversely effected by both tumor development and cytotoxic chemotherapy. Tumor influenced immune alterations have been demonstrated in humans bearing prostatic cancers and in animals with the Dunning R3327 prostatic adenocarcinoma. We report a sequential analysis
which kinetically demonstrates the effects of tumor development and chemotherapy upon immune markers. Such analysis identifies effective doses and cycles of cytotoxic drugs effective against tumors, yet having effects upon the im-
mune system.
Cyclophosphamide (CTX) was chosen because at
low doses it synergistically interacts with the immune sy-
stem. Rats bearing or not bearing the R3327 MAT-LyLu variant of the Dunning tumor were evaluated for immune markers
following tumor implantation and CTX therapy. Splenic leukocytes were assayed for T cell subsets with monoclonal antibodies and adherent cell production of E series prostaglandins (PGE) by thin layer chromatography. RESULTS Time elapsed %Helper T %Suppressor T H/S %Total T %Monocytes Day O (Normal) 54.7 46.0 1.20 76.7 22,9 Day 5 37.6 33.8 1.11 72.4 19.4 Day 19-1 X CTX 35.0 33.8 1.04 68.8 20.0 Day 26-2 X CTX 36.3 31.3 1.17 67.D 19.0 Tumor produced alterations of T cell subsets are complete
5 days following implantation. A single CTX dose (3Dmg/Kg), further suppressed the helper/suppressor (H/S) T cell ratio, but a second dose, one week later restored a normal H/S
ratio, despite reductions CTX produced in each subset. CTX produced dose dependent reductions in Total, Helper and Suppressor T cell subsets in both normal and tumor bearing animals. Monocytic representation is uneffected by CTX and PGE production (immunosuppressive) by adherent spleen cells was not lowered. Presumably CTX synergy with the immune system acts at different levels.
115A