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accumulation of hemoglobin (in benzidine-reactive colo- nies), was assayed as previously described (5). Isolation of VC-resistant Ri cell lines. Cell line Rl[VCR]a.
MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3535-3540 0270-7306/90/073535-06$02.00/0 Copyright X 1990, American Society for Microbiology

Vol. 10, No. 7

Conversion of Differentiation Inducer Resistance to Differentiation Inducer Sensitivity in Erythroleukemia Cells J. MICHAELI,1 Y. B. LEBEDEV,1 V. M. RICHON,1 Z.-X. CHEN,lt P. A. MARKS,"2 AND R. A. RIFKIND1.2* DeWitt Wallace Research Laboratory, Memorial Sloan-Kettering Cancer Center,l* and The Graduate School of Medical Sciences of Cornell University,2 1275 York Avenue, New York, New York 10021 Received 17 November 1989/Accepted 9 April 1990

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of murine erythroleukemia cells (MELC). Commitment, the irreversible initiation of the program of terminal-cell differentiation, is first detected in HMBA-sensitive DS19-SC9 MELC in culture after 10 to 12 h of exposure to HMBA. Vincristine (VC)-resistant MELC derived from the DS19-SC9 MELC line display increased sensitivity to HMBA and become committed with little or no latent period. In the present study, we showed that the MELC line Rl, which is resistant to HMBA-mediated differentiation, became sensitive to inducer if selected for a low level of VC resistance (95% compared with inhibition in cells cultured without cycloheximide. After 3.5 h, HMBA was added to each culture, and cultures continued in the presence of both cycloheximide and HMBA for an additional 8 h, at which time a sample (105 cells) of each culture was removed and assayed for commitment. The remainder of the cells was washed and resuspended in medium with HMBA. Cultures were assayed for commitment at 24 and 48 h following the initiation of the following cultures with HMBA: (i) DS19-SC9 cultured with 5 mM HMBA (0) or with 5 mM HMBA and 1.5 ,ug of cycloheximide per ml (0) for 11.5 h, as described above; (ii) Rl[VCR]a cultured with 3 mM HMBA (U) or with 3 mM HMBA and 1.5 ,ug of cycloheximide per ml (Ol) for 11.5 h; and DS19[VCR]C15 cultured with 3 mM HMBA (A) or with 3 mM HMBA and 1.5 ,ug of cycloheximide per ml (A) for 11.5 h.

Effects of verapamil on VC resistance and induced differentiation of Rl[VCR]a cells. Resistance to the cytotoxic effect of VC can be reversed by exposure to the calcium channel blocker verapamil (25). To determine whether verapamil could reverse the VC resistance of Rl[VCR]a cells and whether the reestablishment of VC sensitivity affected the response to HMBA, verapamil was added to cultures of Rl[VCR]a cells without and with HMBA or VC. Verapamil (3 ,ug/ml) had little or no effect on the rate of growth of Rl[VCR]a cells, but there was marked inhibition of Rl[VCR]a cell growth when these cells were exposed to both verapamil and VC together (Fig. 4A). Exposure of Rl[VCR]a cells to verapamil and HMBA for as long as 18 h followed by the addition of 5 ng of VC per ml resulted in marked cell growth inhibition (Fig. 4B, curve 2). This suggests that verapamil can effectively suppress resistance of Rl[VCR]a cells for at least 18 h but that it has no effect on the accelerated kinetics of HMBA-mediated commitment of these MELC (Fig. 5). Verapamil had no effect on the growth or commitment of DS19-SC9 or Rl cells (data not shown). P-glycoprotein levels in Rl[VCR]a cells. The overexpression of a 140-kDa plasma membrane P-glycoprotein is associated with resistance to a number of cytotoxic agents, including VC, in cells expressing the so-called multidrug resistance phenotype (1, 8). Rl[VCR]a cells do not overexpress the 140-kDa P-glycoprotein; the level of this protein is similar to that in the parental Rl cells (Fig. 6). The levels of P-glycoprotein in both Rl and R1[VCR] cells may be slightly higher than that in DS19-SC9 cells, but they are distinctly lower than that in cell line V3.17(200), deliberately made highly VC resistant and clearly overexpressing the P-glycoprotein.

72 48 Time in Culture (hrs) FIG. 4. Effect of verapamil on VC sensitivity of Rl[VCR]a cells. (A) Rl[VCR]a cells were grown continuously in the presence of 5 ng of VC per ml (0), 3 mM HMBA (0), 3 ,ug of verapamil per ml (A), or 3 ,ug of verapamil per ml-3 mM HMBA-5 ng of VC per ml (A), and cell growth was determined over 72 h. (B) Rl[VCR]a cells were grown in verapamil (3 pg/ml) and HMBA (3 mM) (O); samples from this culture were taken, and 5 ng of VC per ml was added at the times indicated by the arrows (at 12, 18, and 24 h [curves 1, 2, and 3, respectively]). Cell growth for each of these subcultures (A) was determined at 48 and 72 h.

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DISCUSSION are induced by HMBA to terminal DS19-SC9 cells MELC erythroid differentiation (21). MELC line Rl, derived from DS19-SC9, is resistant to HMBA-mediated differentiation (14). Other MELC lines, selected for resistance to low concentrations of VC, display increased sensitivity to HMBA and are induced with little or no latent period (15). In this study, we demonstrated that several VC-resistant cell lines (designated Rl[VCR]a through d) derived from HMBA-resistant Rl MELC became highly responsive to HMBA. R1[VCR] cells were induced with little or no latent period and showed increased sensitivity to HMBA on a molar basis, as do other VC-resistant MELC lines (15). This suggests that the differentiation-accelerating effect of VC resistance is dominant over differentiation resistance. Rl[VCR]a cells are also relatively resistant to the inhibitory effects of TPA and DEX on HMBA-induced differentiation. Unlike the situation with VC-sensitive DS19-SC9 MELC (11), inhibition of protein synthesis during the early hours of culture of Rl[VCR]a cells with HMBA does not delay the onset of commitment to terminal differentiation. 100 r 80 0)

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Time in Culture(hrs) FIG. 5. Effect of verapamil on the commitment of R1[VCR]a cells grown with 3 mM HMBA (0) or with 3 mM HMBA and 3 jig of verapamil per ml (0).

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CONVERSION OF RESISTANCE TO DIFFERENTIATION INDUCERS 1

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FIG. 6. P-glycoprotein expression. Lanes: 1, parental VC-sensitive DS19-SC9 cells; 2, HMBA-resistant Rl cells; 3, Rl[VCR]a cells; and 4, V3.17(200) (a VC-resistant MELC overexpressing the P-glycoprotein). Western blot (immunoblot) analysis was performed on 100 ,ug of partially purified membrane protein, as described in Materials and Methods. Protein molecular masses are shown at the left.

While Ri MELC are resistant to HMBA-mediated terminal differentiation, HMBA does cause a number of changes which also occur, prior to commitment, during the initial period of inducer-mediated differentiation of the HMBAsensitive parental cell line DS19-SC9 (14, 22). For example, we previously reported that as it does in DS19-SC9 cells, HMBA induces Ri cells to accumulate the chromatin-associated protein H1l (14) and to suppress accumulation of c-myc mRNA and c-myc protein (V. M. Richon, J. Michaeli, Z.-X. Chen, R. A. Rifkind, and P. A. Marks, Abstr. Proc. Am. Assoc. Cancer Res., 1989, 30:430). HMBA does not activate transcription of the globin genes or induce changes in chromatin structure at the al and pmai globin gene domains; both of these events occur later in the induction process in DS19-SC9 cells (23). These observations suggest that the block in Ri cells to inducer-mediated differentiation occurs during the latent period. In R1[VCR] cells, this blockade is bypassed, as are the target sites for TPA- and DEX-mediated inhibition of induced differentiation. This apparent developmental bypass in VC-resistant MELC could be due to the constitutive expression of a protein which is required for commitment and is induced by HMBA during the latent period of VC-sensitive cells. This hypothesis is supported by our finding that inhibition of protein synthesis did not delay commitment of R1[VCR]a or other VC-resistant MELC but did delay commitment of VCsensitive lines. The nature of the relationship between increased responsiveness to induction of terminal differentiation by HMBA and resistance to VC is not defined. Verapamil restores the sensitivity of Rl[VCR]a cells to VC, presumably by allowing increased accumulation of VC, as it does in other VCresistant cells (25), but verapamil does not alter the sensitivity of VC-resistant MELC to induction by HMBA. Thus, the increased sensitivity to HMBA that is characteristic of the several independently derived VC-resistant cell lines does not appear to depend on that function of the drug transport channel blocked by verapamil. Indeed, while VC-resistant Ri cells do exhibit some features of the multidrug resistance phenotype (3, 7, 8, 19, 25), namely, a pattern of cross resistance to cytotoxic agents, reduced accumulation of the vinca alkaloid, and suppression of resistance by verapamil, there is no detectable overexpression of the 140-kDa membrane-associated P-glycoprotein compared with the level of expression in the parental Ri cells. The significance of the slightly elevated expression of the P-glycoprotein in both Ri and Rl[VCR]a cells, compared with that in DS19-SC9, is

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unclear. The facts that HMBA inhibits Rl cell growth in a dose-dependent manner, as it does for DS19-SC9 cells (J. Michaeli, unpublished observation), and that verapamil does not effect the inducibility of either Rl or R1[VCR] cells suggest that these small differences in P-glycoprotein expression do not account for the differentiation resistance of Rl cells. The VC resistance of Rl[VCR]a cells probably involves a mechanism of drug resistance that is independent of P-glycoprotein and the mdr genes, and the relationship of this alternative resistance mechanism to HMBA sensitivity has yet to be determined. We have previously reported that MELC, including Rl and R1[VCR] cells, have both PKCot and PKC, activity, and in each, PKCa is the principle isoform (17). However, the fraction contributed by PKC, differs between these cell lines. In general, MELC variants which are relatively resistant to induction are also relatively low in PKCP activity, while those with an accelerated response display higher PKC, activity (17). The level of PKCa is fairly constant among the MELC tested. The level of PKC, in Rl[VCR]a cells is 4-fold that in Rl cells and about 2.5-fold that in DS19-SC9 cells (17). A role for PKC activity during induction is further supported by the observation that depletion of PKC activity by exposure to TPA inhibits HMBA-mediated induction (16). Even VC-resistant MELC, which are relatively resistant to inhibition of differentiation by TPA, can be depleted of PKC and inhibited with respect to differentiation if they are preincubated with TPA for about 6 h (17). On the basis of these observations, one can speculate that VC resistance is associated with elevated levels of PKCB activity and that this, in turn, may account for increased sensitivity to HMBA. One effect of HMBA during induction appears to involve translocation and activation of PKC at the plasma membrane (unpublished observation). The phosphorylation target of this PKC activity is unknown, but it may include the protein that is constitutively expressed in VC-resistant MELC or other proteins, such as members of the family of cell cycle-regulating proteins (4, 18). Another implication of the present findings is that clinically acquired resistance to differentiation inducers might be overcome by exposure to a cytotoxic agent, i.e., VC. This has possible implications for the design and implementation of trials of differentiation inducers in the treatment of cancer. ACKNOWLEDGMENTS

This work was supported in part by Public Health Service grant CA-31768 from the National Cancer Institute and by grants from the Roberta C. Rudin Leukemia Research Fund and the Japanese Foundation for Promotion of Cancer Research. Zi-Xing Chen was supported by a Rockefeller Foundation Biotechnology Career Fellowship. LITERATURE CITED 1. Burnette, W. N. 1981. "Western blotting." Electrophoretic

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