Inhibition of Antibody-Complement-Mediated Killing of Tumor Cells by Hormones1

[CANCER RESEARCH 36, 3672-3677, October 1976] Inhibition of Antibody-Complement-mediated Killing of Tumor Cells by Hormones1

SeymourI. Schlager, Sarkis H. Ohanlan,and Tibor Borsos Biology Branch,NationalCancerInstitute, NIH, Bethesda,Maryland2XKl14

SUMMARY chemotherapeutic agents (30, 31), or proteolytic enzymes (6) renders the cells susceptible or increases their sus Line 1, a chemically induced guinea pig hepatoma, is ceptibility to killing by antibody and GPC. It was concluded susceptible to killing by anti-Forssman immunogbobulin M from these studies that resistance to cytotoxicity may be antibody and guinea pig complement. When these tumor due to intrinsic properties of the cells to prevent comple cells are pretreated at 37Â°with 10@ to 10â€•M concentra ment-mediated damage to their surface (5, 6, 29-32). tions of the polypeptide hormone insulin, with the catechol Antibody-complement-mediated attack results in mor amine L-epinephrine-HCI, or with the glucocorticoid ste phological and chemical changes of the cell membrane roids hydrocortisone sodium succinate or prednisobone so (15). Since steroid and polypeptide hormones have been dium succinate, the cells show a marked reduction in their shown to increase the stability and/or to decrease the susceptibility to killing by antibody and guinea pig comple permeability of cell membranes (1, 2, 10, 14, 16, 19, 22, 36, ment; pretreatment at 0Â°isineffective. Similar results were 38-40), a number of these hormones were examined for obtained with another antigenically distinct guinea pig hep their ability to modify the effects ofin vitro humoral immune atoma (line 10) when tested with anti-Forssman immuno attack. The purpose of this paper is to document the various globulin M or specific antitumor antibodies and human aspects of hormone treatment of tumor cells and to show complement. The ability of the hormones to render the cells that, following treatment with certain hormones, the cells resistant is dependent on time, temperature, and hormone are less susceptible to killing by antibody and complement. concentration . The effect of hormone treatment is maximal between 30 and 60 mm and is reversible within 4 hr even in the continued presence of hormone. Treatment of line 1 MATERIALS AND METHODS cells with up to 10,000-fold greater concentrations of the less biologically active or inactive analogs, DL-epinephrine, Animals. Inbred Sewall-Wright strain 2 male guinea pigs /3-estradiol, testosterone, or proinsulin has no effect on the were obtained from the Frederick Cancer Research Center, susceptibility of the cells to killing by antibody and guinea Frederick, Md. The animals weighed approximately 250g. pig complement. The effect of hormone treatment is not Cells and Reagents. Sheep erythrocytes were collected due to a direct inactivation of bound or fluid-phase comple and washed as previously described (26). ment components by the hormones or to a decrease in the Two antigenically distinct diethylnitrosammne-inducedhe ability of the cells to bind complement-fixing antibody. patic tumors (ascites form), line 1 and line 10 passed in strain 2 guinea pigs, were collected as described (25, 42, 43) and suspended in RPMI 1640-20% FCS. All cytotoxicity INTRODUCTION assays were performed in VBS-gel. The hormones used in these studies were: hydrocortisone The dieth@Initrosamine-induced guinea pig hepatoma, sodium succinate (Upjohn Co., Kalamazoo, Mich.; Lot 343- designated line 1, is susceptible to killing by anti-Forssman BR); prednisolone sodium succinate (National Cancer Insti antibody and GPC2 but not by specific antitumor antibody tute, Cancer Therapy Evaluation Branch, Bethesda, Md.; and GPC (24). The antigenically distinct line 10 hepatoma, NSC 9151); bovine insulin (Sigma Chemical Co., St. Louis, when sensitized with either antibody, is susceptible to Mo., Lot 24C-3130; 24.3 lU/mg); L-epinephrine methyl ether killing by HUC but not by GPC (24). Pretreatment of the HCI (Sigma; Lot 84B-5140); DL-epinephrine (Sigma; Lot tumor cells with certain metabolic inhibitors (29, 31), 650-001 1); @1.3,5(â€˜Â°@-estratrien-3,17-f3-diol(f3-estradiol; Sigma; Lot 45C-O100);i@4-androsten-17f3-oI-3-one(testoster I This is Paper 8 of the series, â€œLysis of Tumor Cells by Antibody and one; Sigma; Lot 113C-1460); pork insulin, chicken insulin, Complement.â€•Presented in part at the 67th Annual Meeting of the American Association for Cancer Research, May 8. 1976, Toronto, Ontario, Canada pork proinsulin, pork DAA-insulin and the A and B chains of (28). pork insulin (courtesy of Dr. Pierre De Meyts, Clinical Endo 2 The abbreviations used are: GPC, guinea pig complement; HUC. human crinobogy Branch, National Institute of Arthritis, Metabolism complement; GIBCO, Grand Island Biological Co., Grand Island. N. V.; RPMI 1640-20% FCS, Roswell Park Memorial Institute tissue culture Medium 1640 and Digestive Diseases, NIH). (GIBCO No. 187G) containing 20% fetal calf serum (GIBCO No. 614) and 1% TreatmentofTumorCellswithHormones.Unlessother antibiotic-antimycotic mixture (GIBCO No. 524); VB5-gel, isotonic Veronal wise specified, tumor cells (5 x lO@@cells/mI)were cultured buffered NaCI solution containing 0.1% gelatin, 0.001 N Mg2@,and0.00015 IA Ca2@;DAA,desalanine-desasparagine. in 10-mI volumes of RPMI 1640-20% FCS with the appropri Received April 7. 1976; accepted June 16. 1976. ate concentrations of hormone in 100- x 15-mm plastic

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. Hormones and Antibody-Comp!ement Killing bacteriological Petri dishes (Falcon Plastics, Oxnard, Calif.) at 37Â°under 5% CO2-air. After incubation, aliquots of the cell cultures were drawn off, dispensed into 12- x 75-mm polypropylene capped tubes (No. 2063; Falcon), washed 5 times in VBS-gel, and tested for their susceptibility to killing by antibody and w complement. All cells that were to be reincubated in culture :, -J medium without added hormone were first washed, trans m ferred to a new set of 12- x 75-mm capped tubes, and z resuspended in fresh RPMI 1640-20% FCS. >- Sera and Antisera. RabbitantiserumtosheepForssman I- antigen was prepared as described previously (26). Ten ml I I- were heated for 30 mm at 56Â°andpassed through a column of Sephadex G-200. The 1gM-rich fraction was collected, w pooled, and stored at â€”20Â°untilused. z Tumor-specific rabbit anti-line 1 and anti-line 10 antisera were prepared as previously described (4). The antisera Cl) U) -J were heated for 30 mm at 56Â°,absorbed with sheep erythro -J w cytes and with the appropriate tumor cells, filtered, and 0 stored at â€”20Â°. Normal GPC (JEM Research Products, Inc., Kensington, Md.) and normal HUC, absorbed at 0Â°with sheep erythro cytes and line 1 and line 10 tumor cells, were stored at â€”30Â°. Antibody-Complement-mediatedCytotoxicityof Tumor Cells. This method for determining antibody-complement mediated killing has been described (25). Briefly, 0.1 ml of diluted antiserum was added to 0.1 ml of tumor cells (106/ 1 2 4 1 2 4 ml). After incubation for 30 mm at 30Â°thecells were washed RELATIVE ANTiBODY CONCENTRATiON with VBS-gel and incubated with 0.1 ml GPC (diluted 1:8) or Chart 1. Effect of anti-Forssman gM antibody concentration (1 = 1:20) on HUC (diluted 1:10) for 60 mm at 37Â°.One-tenth ml of 0.4% the killing of hormone-treated line 1 cells by GPC diluted 1:8. A, L-epifleph trypan blue was added, and those cells taking up the dye rine-HCI: â€¢,5X 10-i IA; A, 5.5 x 10@IA; @,5.5x 10@ N. B, hydrocortisone sodium succinete: â€¢,10' IA; A, 10@M; â€¢,10@M. C, insulin; â€¢,10@IA; A, were counted visually. Controls included cells plus comple 10-i IA;U, 10' IA. 0, pradnisolone sodium succinate; â€¢,7.5X 10 IA; A, ment alone and cells plus buffer alone. The error of the test 10-i M; U, 10 IA. 0, line 1 cells not treated with hormone. Date represent3 is less than Â±10%. separate experiments showing spread of values obtained. Values have been corrected for complement controls (line 1 cells plus GPC alone).

RESULTS specific anti-line 1 antibody (data not shown). Hormone treated line 10 cells also showed a marked increase in Antibody-Complement-mediatedCytotoxicity of Hor resistance to killing by either anti-Forssman or specific anti mone-treated Tumor Cells. Five million line 1 tumor cells in line 10 antibodies and HUG, compared with untreated cells 10 ml RPMI 1640-20% FCS, containing various concentra (data not shown). In all cases with both tumor cell lines, tions of L-epinephrine-HCI, insulin, prednisobone sodium complement control and cell control values remained below succinate, or hydrocortisone sodium succinate, were incu 10%. Since the overall effect of the hormones on the line 1 bated at 37Â°under air-5% CO2. Control cultures were pre and line 10 cells was similar regardless of the antibody or pared concurrently with cells in tissue culture medium complement source used, we used line 1 cells treated with alone. After 30 mm, the cells were washed 3 times with 10 anti-Forssman 1gM antibody and GPC as the system for all ml RMPI 1640-20% FCS and 2 times with 10 ml VBS-gel, further experiments. resuspended to 10@cells/mI in VBS-gel, and assayed for Effect of Hormone Concentration in Decreasing the Sen their susceptibility to killing by anti-Forssman antibody and sitivity of Tumor Cells to Anflbody-GPC-mediated Killing. GPC. Chart 1 illustrates the combined results of 3 experi Dose-response experiments were performed to determine ments. Hormone-treated line 1 cells were less susceptible the optimal concentration of each hormone that was most than were the control cells to killing by anti-Forssman anti effective in rendering the line 1 tumor cells resistant to body and GPC. antibody- and GPC-mediated killing. Serial 100-fold dilu Resistance to killing by GPC was observed with cells tions of hormones between 10@ and 1015 M were tested in coated with limiting, near excess, or excess amounts of 5-mI volumes of RPMI 1640-20% FCS, containing 5 x 10@ anti-Forssman antibody (Chart 1). Similarly, hormone line 1 cells/mI. The cells were cultured for 30 mm at 37Â°, treated line 1 cells, which are more susceptible to killing by washed, and examined for their susceptibility to killing by HUC when coated with antibody, demonstrated increased antibody and GPC. The results of a representative experi resistance to killing. However, this occurred only when the ment are presented in Chart 2. Line 1 cells, incubated with cells were coated with limiting dilutions of anti-Forssman or concentrations of L-epinephrine-HCI, hydrocortisone, or

OCTOBER1976 3673

S. I. Schiager et a!.

to the loss of hormone activity. Hormones incubated for 4 hr at 37Â°in RPMI 1640-20% FCS alone or supernatant fluids from 4-hr cultures of line 1 cells plus hormone were still capable of rendering fresh line 1 cells resistant to killing by antibody and complement. The reversion of line 1 cells to the susceptible state in the

Lu continued presence of hormone was now contrasted with @1 their reversion when washed free of hormone. Line 1 cells (5 z x 105/ml) were incubated in RPMI 1640-20% FCS with hor

>. mones for 30 mm at 37Â°.The cells were then washed 10 times with 10 ml of RPMI 1640-20% FCS and resuspended at I 5 x 10@cells/mI in RPMI 1640-20% FCS without added hormone. The cells were tested for susceptibility to killing 0Lu z immediately and after incubation for an additional 30, 60,

I- 120, and 240 mm at 37Â°.Theresults in Chart 4 show that the U) cells remained resistant to killing for up to 60 mm at 37Â° U) -J Lu-I after transfer to hormone-free medium but reverted to the C., sensitive state within 2 hr. The reversion to the sensitive state could not be prevented by culturing the washed cells at 0Â°. Effectof Temperatureon HormoneTreatmentof Tumor Cells. Line 1 tumor cells were cultured at 0Â°or37Â°in RPMI 1640-20% FCS with and without hormones. After 30-, 60-, and 120-mm incubations,thecellswerewashed and tested for their susceptibility to killing by antibody and GPC. Table i07 10' i@-@ iO-@ Not Treated HORMONECONCENTRATION(molar) witt@ en Honnone AB:@- Chart 2. Effect of hormone concentration on the susceptibility to killing of hormone-treated line 1 cells by antibody and GPC. A, L-epiflephrifle-HCI; B, insulin; C, prednisolone sodium succinate; 0, hydrocortisone sodium succi nate;U,cellsplusanti-Forssman1gMantibodydiluted1:10plusGPCdiluted 1:8; @,cellsplus GPC diluted 1:8; 0, cells plus buffer alone. w prednisolone as low as 10'@ M, were markedly depressed in -I their susceptibility to killing. Insulin was effective at con z

centrations as low as 10_13M (Chart 2). >. The steroid hormones at concentrations of 10@ M or I-. â€˜ @_@_@_---1i@ greater were toxic, whereas neither of the polypeptide hor I I- mones was toxic at 10@M, the highest concentration tested (Chart 2). On the basis of these results we concluded that w the lowest concentrations of hormone resulting in the maxi z mal effect on the cells were 10@ M hydrocortisone, epi nephrine, and insulin and 10@ M prednisobone. All further U) U) experiments were performed with these concentrations. -J w-J Effect of Time of HormoneTreatment of Tumor Cells. 0 Line 1 tumor cells were cultured in RPMI 1640-20% FCS with and without hormones. After 15, 30, 60, 120, and 240 mm of incubation, aliquots of the cell suspensions were removed and the cells were washed and examined for their susceptibility to killing by antibody and GPC. Chart 3 illus trates that the hormone-treated cells begin to show resist ance to killing within 15 mm and that maximal resistance is 153060 120 240 153060 120 240 reached within 30 to 60 mm in culture. After 60 mm of incubation, the cells begin to revert to their sensitive state TIMEOFINCUBATIONWITHHORMONE(MIN) and are indistinguishable from control cells within 4 hr Chart 3. Effect of time of incubation of line 1 cells with hormone on the (Chart 3). Ce!l control (line 1 cells alone) and complement susceptibility of the cells to killing by anti-Forssman 1gMantibody (1:10) and GPC (1:8).A, 10' IAL-epinephrine-HCI; B, 10@IAinsulin; c, io-@IAhydrocor control (cells plus GPC alone) values remained less than tisone sodium succinate; D, 10' IA prednisolone sodium succinate. â€¢, 10% throughout the experiments. hormone-treated cells; 0, complement control values (line 1 cells plus GPC alone) for hormone-treated cells; A, line 1 cells not treated with hormone The reversion of the line 1 cells to a susceptible state in (values corrected for complement control). Values represent mean Â±S.D.for the continued presence of hormone could not be attributed 3 separate experiments.

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1 shows that cells cultured with hormone at 0Â°foras long as to killing after 30 to 60 mm and began to revert to their 2 hr remained as sensitive to killing as the untreated con susceptible state within 2 hr (Table 1). trols. Subsequent experiments have shown that line 1 cells Effectof Less PhysiologicallyActiveHormoneAnalogs cultured with hormones at 0Â°for as long as 4 hr remain on Tumor Cell Killing. For each hormone used in this study, susceptible to killing (data not shown). As expected, cells there exists an analog that physiologically is either less cultured at 37Â°withthese hormones were rendered resistant active or inactive (10, 23, 27, 36). The analogs of L-epineph rine HCI (DL-epinephrine) and of hydrocortisone succinate and prednisolone succinate (f3-estradiol and testosterone) were tested for their effect on the susceptibility of line 1 cells to killing by antibody and GPC. Line 1 cells were incubated in RPMI 1640-20% FCS at 37Â°with 10@ M L epinephrine-HCI, with 10@ M hydrocortisone sodium succi nate, or with 100- or 10,000-fold higher concentrations of LU their less active analogs. Controls included cells in RPMI :D -J 1640-20% FCS without added hormone. After 30-mm, 2-hr, z 4-hr, and 17-hr incubations, aliquots of the cell suspensions were collected and the cells were washed and tested for >. their sensitivity to killing by antibody and GPC. Table 2 I- I :@ shows that only those cells cultured in L-epinephrine-HCI or hydrocortisone were rendered resistant to killing. At the times studied, no increase in resistance to killing by anti 0 LU z body and GPC was observed with cells cultured in medium containing the hormone analogs (Table 2). I-. U) The results obtained with insulin from 3 species (bovine, U) -J chicken, and pig), the less physiologically active analogs of -J LU pork insulin (DAA-insulin and proinsulmn), and the isolated A 0 and B chains of pork insulin are presented in Table 3. Bovine, chicken, and pork insulins were all effective in rendering the line 1 cells resistant to killing by anti-Forss man antibody and GPC (Table 3). Pork DAA-insulin at 10@M was marginally effective in rendering the cells resistant; pork proinsulin at 10@ M and the isolated insulin A and B : chains at 106 M were ineffective (Table 3). 03060 120 240 03060 120 240

TIMEOFINCUBATIONWITHOUTHORMONE(MIN) DISCUSSION Chart 4. Sensitivity of line 1 cells, pretreated with hormone for 30 mm at 37â€•andthen recultured at 37Â°inthe absence of hormone, to killing by anti Forssman 1gM antibody (1:10) and GPC (1:8). A, pretreated with 10@ IA These studies are a continuation of efforts to analyze the epinephrlne HCI; B, pretreated with 10@ IAinsulin; C, pratreated with 10@N interaction of tumor cells with antibody and complement (5, hydrocortisone sodium succinate; 0, pretreated with 10' N prednisolone 8, 9, 24, 25, 29-32). The data presented in this paper show sodium succinate. â€¢,hormone-treated cells; 0, complement control values (line 1 cells plus GPC alone) for hormone-treated cells; A, line 1 cells not that line 1 tumor cells incubated in medium containing the treatedwithhormone(correctedforcomplementcontrolvalues). polypeptide hormone, insulin, the catecholamine L-epi

Table 1 Effect of temperaturecomplementHormone%on the abilityof hormones to renderline 1 cells resistant to killing by antibody and blue3Omina60mmâ€•120cells stained with trypan mirY'0Â°37Â°0Â° 370concen 37Â°0@ TAGTCL-Epinephrine-HCI10@51119838Treatmenttration (M)TAC@TCTACTCTAC TC TAC TCTACTC 351Hydrocortisoneso 5 16 4634 272dium 10@45516039 6 11 2591 succinateInsulin10@45312440 304Prednisolone 1 7 0721 261succinateNone481049542sodium10@39416746 5 13 2555 4 41 2655 484 a Time of incubation of line 1 cells with hormone. b TAC, antibody-coated line 1 cells plus GPC (anti-Forssman 1gM 1 :10 pIus GPC 1 :8); TC, complement control (line 1 cells plus GPC). Cell controls (line 1 cells alone) also remainedbelow 10%.

OCTOBER 1976 3675

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1976 American Association for Cancer Research. S. I. Sch!ager et a!. Table 2 Effectof lessactiveor inactivehormoneanalogson antibody-complement-mediatedkillingof line 1 cells % cells stained with trypan blue

30 mm0 120 mmâ€• 240 mm0 17 hr0

Treatment10-i TAGâ€• TG TAG TC TAG TG TAG TC ML-epinephrine-HGI8425540543210@5 NDL-epinephrine49357642343310@ MDL-epinephrine414444401481010@ 222312436434diumM hydrocortisone so succinate10-s IAfJ-estradiol44340435462510@ IAfl-estradiol48343135653710@ IAtestosterone48249231338110@ Mtestosterone5565865735921None374502412444

a Time of incubation of line 1 cells with hormone. b TAG, antibody-coated line 1 cells plus GPC (anti-Forssman 1gM 1 :10 plus GPC 1 :8); TG, complement control (line 1 cells plus GPC).

Table 3 hormones for prolonged periods of time; (c) failure to dem Effectof insulin obtained from severalspecies,insulin analogs,and onstrate the kinetics of cell-hormone interaction; (d) lack of insulin components on antibody-complement-mediatedkilling of a well-defined assay system with appropriate controls; (e) line 1 cells lack of a well defined antibody and complement source. trypanTreatmentblueTAO'TC10-i % cells stained with In our studies, we have attempted to avoid these prob lems and work within a well-defined system. It is apparent from this study that, while hormone concentration is impor insulin151210.-aM bovine tant, the length of time for which the cells are exposed to insulin17810â€”aIAbovine the hormone is also critical. In our system, the tumor cells insulin12610-11M chicken showed maximal resistance within 30 to 60 mm of exposure insulin20610-iM chicken to the hormones and reverted to the sensitive state within 4 insulin27910-sIApork hr. Furthermore, resistance by the cells to killing was ob proinsulin49310-iM pork proinsulin45510-iM pork served at 37Â°but not at 0Â°,suggesting that conditions nec DAA-insulin291110-aIApork essary for hormone binding to the cell and for cell metabo A-chain54510-aM pork insulin lism may be necessary for the expression of the resistant B-chain4911None4010IA pork insulin state. The action of the hormones on the line 1 cells is not due to a decrease in the amount of detectable Forssman or a TAC, antibody-coated line 1 cells plus GPC (anti-Forssman 1gM tumor-specific antigen on the cell surface. According to the 1:10 plus GPC 1:8); TG, complement control (line 1 cells plus GPC). Cl fixation and transfer test (26), hormone-treated line 1 nephrine-HCI, or the glucocorticoid steroids hydrocortisone cells that were resistant to killing by antibody and GPC sodium succinate or prednisolone sodium succinate were could bind as much Cl-fixing anti-Forssman or specific rendered resistant to killing by anti-Forssman 1gMantibody antitumor antibody as could untreated control cells (S. I. and GPC. This effect was dependent on hormone concen Schlager, unpublished Qbservation). tration, temperature, and time and was reversible. Similar It has been proposed that the observed resistance of results were observed with line 10 and line 1 cells under erythrocytes to lysis by antibody and complement may be attack by anti-Forssman or specific antitumor antibodies due to a direct anticomplementary effect by high concentra and HUC. Tumor cells cultured with less physiologically tions of hydrocortisone on fluid-phase complement when active analogs of the hormones were not rendered resistant. the complement source is added to the cells with the hor There have been reports that hormones may inhibit (12, mone (13). In this report, however, an inhibitory effect by 17, 18, 21, 36, 37), enhance (11), or have no effect on the hormones on fluid phase complement is unlikely. GPC humoral immune attack (20, 33, 34). These apparent contra diluted in any of the 4 hormones used in our studies killed dictions may be due to variations in the amounts of hor antibody-coated line 1 cells to the same degree as did GPC mones used in these investigations, to differing experimen diluted in hormone-free media. In addition, the 4 hormones tal designs, or to the target cells used. In addition, these used were effective at concentrations ranging from l0@ to reports are difficult to evaluate because they suffer from 1 approximately 10@ M; this is within the detectable physio or more of the following deficiencies : (a) the use of high logical range of concentrations for these hormones (10, 35). concentrations of hormones (10@ to 10@ M) that do not Finally, the tumor cells were routinely washed to remove reflect physiological conditions prevailing in the cells and unbound hormone prior to testing for susceptibility to kill may be anticomplementary; (b) incubation of cells with the ing.

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To test the effect of hormone treatment on membrane 18. Jennings, J. F., and Taylor, G. Effect of Hydrocortisone Hemi-Succinate bound complement components, the hormones were added on Immune Lysis of Sheep Erythrocytes. Nature, 203: 661, 1964. 19. Lacko, L., Wittkee, B., and Geck, P. Interaction of Steroids with the to line 1 cells in the T* state. The T0state is an intermediate Transport System of Glucose in Human Erythrocytes. J. Cellular Phys in which the tumor cell has at least 1 membrane-bound iol., 86: 673â€”680,1975. 20. Letta, H., and Kutsakis, A. Gytotoxic Effects of Specific Antiserum and antigen-antibody complex and all the necessary comple 17-Hydroxycorticosterone on Cells in Tissue Culture. Lab. Invest., 6: 12- ment components bound for killing to occur (7). Preliminary 27, 1957. experiments indicated that the hormones reversibly inhibit 21. Lee, K. C., Langman, R. E., Paetkau, V. H., and Diener, E. The Cellular Basis of Cortisone-induced Immunosupprassion of the Antibody Re the transformation of line 1 cells in the T* state to dead cells. sponse Studied by its Reversal In Vitro. Cellular Immunol., 17: 405-417, These results suggest that the hormones do not directly 1975. 22. Libertini, L. H., Waggoner. A. S., Joel, P. C., and Griffith, 0. H. Orients affect membrane-bound complement components. tion of Lipid Spin Labels in Lecithin Multilayers. Proc. NatI. Acad. Sd. U. The mechanism by which hormone treatment decreases S., 64: 13-19, 1969. the sensitivity of the cells to attack by antibody and comple 23. Lippmen, M. E., Heltermen, R. H., Leventhal, B. G., Perry. S., and Thompson, E. B. Glucocorticoid-binding Proteins in Human Acute Lym ment is not understood. It has been shown that hormones phoblastic Leukemia Blast Cells. J. Glin. Invest.. 52: 1715-1725. 1973. (especially the glucocorticoid steroids) increase the stability 24. Ohanian, S. H., and Borsos, T. Lysis of Tumor Cells by Antibody and and integrity of cell membranes (1, 3, 14, 16, 17, 19, 22, 33, Complement.II. Lack of CorrelationBetweenAmount of G4 and C3 Fixed and Cell Lysis. J. Immunol., 114: 1292-1295, 1975. 35, 36, 38, 39, 41, 42). This may occur by direct interaction 25. Ohanian, 5. H., Borsos, 1., end Rapp, H. J. Lysis of Tumor Cells by of the hormone with a membrane component (2, 22, 39) or Antibody and Complement. I. Lack of Correlation between Antigen Con tent and LyticSusceptibility.J. NetI. Cancer Inst., 50: 1313-1320, 1973. indirectly through specific binding to intracellular hormone 26. Rapp, H. J., and Borsos, T. Molecular Basisof Complement Action, pp. receptors (2, 3). Further studies with the line 1 system offer 75-109. New York: Appleton-Century-Crofts, Inc., 1970. an opportunity to examine these possibilities critically. 27. Rosenau, W., Baxter, J. D., Rousseau, G. G. , and Tompkins, B. M. 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OCTOBER1976 3677

Seymour I. Schlager, Sarkis H. Ohanian and Tibor Borsos

Cancer Res 1976;36:3672-3677.

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