[CANCER RESEARCH 40, 56-60, January 1980] 0008-5472/80/0040-0000$02.0O Direct Determinationof the Influenceof the Cell Cycle on the Sijrvival of Tumor Cells Exposedto Cytotoxic Antibodies' Paul J. Leibson,2 Michael R. Loken, Susan J. Shapiro, and Hans Schreiber@ La Rabida-University of Chicago Institute, Chicago 60649; and Departments of Pediatrics (P. J. L.j, Microbiology (M. R. Li, and Pa@olopy(H. SI, and Committee on Immunology(S. J. S.), University of Chicago, Chicago, Illinois 60637 indicated that cells in the G1phase of@thecell cycle are most susceptible to Iysis by cytotoxic antib4dy. However, there are other studies that have indicated either that G1 cells can be more resistant to antibody (18) or thattthere are no cell cycle specific changes in susceptibility (10)@The major problem in interpreting these results is that all @f the studies involved artificial synchronization or physical 4eparation of the target cells before they were exposed to th4 antibodies. The drugs used to synchronize populations or th4 changes produced by the separation techniques may by then'@selves alter the suscep tibility of the cells to cytotoxic antib@x1ies.Therefore, such studies are not necessarily analogous@tothe effect one might observe when untreated, exponentialI@1growing cells are ex posed to antibody. We report here a method by which @synchronous cell pop ulations exposed to a lethal agent (@,hetherit be cytotoxic antibodies, antineoplastic drugs, or irr@diation)can be directly analyzed inorder to determine howrna@iycells were killed from each phase of the cell cycle. Usin@ exponentially growing murine Si 07 myeloma cells, we found no major loss of cells from one phase of the cell cycle priorj to the death of cells in other phases, and, therefore, there we@eonly small differences in the susceptibility of cells in diffei1@ent phases to specific antibodies. MATERIALS AND METHODS ABSTRACT We have developed a direct method for measuring the influ ence of the cell cycle on the survival of tumor cells exposed to antibody and complement. Asynchronous, exponentially grow ing Si 07 myeloma cells were exposed to antisera reactive with either histocompatibility, viral, or other surface antigens. The resulting mixtures of live and dead cells were simultaneously analyzed for viability and DNA content using a modified fluo rescence-activated cell sorter, which was adjusted to record only the DNA content of the tumor cells surviving antibody treatment. The cell cycle analysis of the tumor cells surviving exposure to either of the three antisera indicated that there was no major loss of cells from a single phase of the cell cycle prior to the death of cells in other phases. The relative risk of cell death was only 1.03 times higher for G1 cells than for either S or 02 + M cells. Even when myeloma cells were exposed to a concentration of antihistocompatibility serum that killed 96% of the cells, there was only a small decrease in the proportion of cells in G1. These studies were extended to populations of cells which had altered sensitivity to cytotoxic antibody after being treated with the chernotherapeutic agents melphalan or actinomycin D. Using the method described above, we demonstrated that the drug-induced changes in susceptibility were not due to the cell cycle effects of the two drugs. The method of analysis described here should also be useful for directly determining how the changes that occur during the cell cycle can affect the susceptibility of tumor cells to other lethal agents. INTRODUCTION The recognition that cells in separate phases of the cell cycle have different functional properties has generated considerable biological and therapeutic interest. Cell cycle-dependent changes in surface antigen expression (5, 8, 12), intracellular metabolism (4, 6, 19), and membrane structure and function (17) have been described. These alterations in phenotypic characteristics can result in differential susceptibility to treat ment by irradiation (22), drugs (25), and immune reagents (7, 15). Specific attention has been directed toward whether there are cell cycle-dependent differences in the susceptibility of cells to cytotoxic antibodies. Several studies (15, 17) have I Supported by USPHS Grant ROi -CA-22677 from the National Cancer lnsti tute and Grant 78-78 from the American Cancer SocIety-Illinois Division. 2 To whom requests for reprints should be addressed, at La Rabida-University of Chicago Institute, East 65th St. and Lake Michigan, Chicago, Ill. 60649. Supported by USPHS Training Grant I-T32-HD-07009 from the National Institute of Child Health and Human Development. 3 Recipient of Research Career Development Award K04-CA-00432. Received July 30, i 979; accepted October 5, 1979. 56 Cells. A freshly recloned SI 07 @ myeloma culture line (Si 07.3.7.1 ) of BALB/c origin was us@din this study. This cell line grows in complete Dulbecco's rt@odifiedEagle's medium with a doubling time of about 15 hr. s@creteshigh amounts of phosphorylcholine-binding myeloma @roteln (dxi and sc),and also displays a high density of surfac4 immunoglobulin (20). It also actively releases a type C retrov@ruswhich has antigenic cross-reactivity with Rauscher murine@ leukemia virus (14). Antisera. Anti-H-2― serum (C57B1 anti-DBA) was the gen erous gift of Dr. Frank Fitch, University of Chicago, Chicago, III. Guinea pig antiserum raised agair@t Rauscher munne leu kemia viruses was obtained from EI4@tro-NucIeonicsLabors tories (Bethesda, Md.), after it had be@nraised against double density gradient-purified Rauscher mtirine leukemia virus iso lated from an infected JLS-V9 mouse (BALB/c) bone marrow cell line. This antiserum (complementfixation titer = 1/80 for purified virus; complement fixation titer <1 /1 0 for fetal bovine serum) has previously been shown to react with myeioma cells because of the antigenic cross-reacti@itybetween the 71 ,000- dalton glycoprotein component of R@uschermurine leukemia virus and the retrovirus released by th@Si 07 myeloma cell line (13, 14). Rabbit antiserum to whole @1 07 myeloma cells was prepared by s.c. immunization with @ O@SI07 myeloma cells emulsified in complete Freund's adju@,ant followed by monthly CANt@ERRESEARCHVOL.40 Research. on February 25, 2016. © 1980 American Association for Cancer cancerres.aacrjournals.org Downloaded from