Hexamethylene bisacetamide induces programmed cell death (apoptosis) and down-regulates BCL-2 expression in human myeloma cells

Abstract

Multiple myeloma (MM) is a B cell malignancy characterized by the expansion of monoclonal Ig-secreting plasma cells with low proliferative activity. It is postulated that inhibition of physiologic cell death is an underlying factor in the pathophysiology of MM. The development of chemoresistance is a common feature in patients with MM. In the present studies, hexamethylene bisacetamide (HMBA), a hybrid polar compound that is a potent inducer of terminal differentiation of various transformed cells, is shown to inhibit the growth of several human myeloma cell lines (ARP-1, U266, and RPMI 8226), including doxorubicin-resistant RPMI 8226 variants that overexpress the multidrug-resistance gene, MDR-1, and its product, p-glycoprotein. In addition to growth arrest and suppression of clonogenicity, HMBA induces apoptosis both in freshly isolated human myeloma cells and in cell lines, as determined by morphologic alterations, cell cycle distribution and endonucleosomal DNA fragmentation. Further, HMBA decreases BCL-2 protein expression in myeloma cells within 12-48 hr. Overexpression of BCL-2 protein in ARP-1 cells confers resistance to HMBA-induced apoptosis. Taken together, these data suggest that HMBA is a potent inducer of apoptosis in human myeloma cells, which may act through suppressing the anti-apoptotic function of the bcl-2 gene. HMBA, and related hybrid polar compounds, may prove useful in the management of this presently incurable disease.

Figure 1

Effect of HMBA on myeloma cell line growth. ARP-1 or U266 cells were incubated at 10⁵ cells per ml in RPMI 1640 with 10% FBS. (A) cells were incubated in the presence of the indicated concentration of HMBA. Viable cell numbers were determined at 72 hr by trypan blue exclusion. Inhibition of cell growth is expressed relative to controls. (B) Cells were cultured in the presence of 5 mM HMBA for the indicated periods of time. Inhibition of cell growth is expressed relative to controls.

Figure 2

Morphological alterations in cells cultured with HMBA. (A) U266 cells cultured without HMBA for 24 hr. (B) U266 cells, cultured with HMBA for 24 hr, display nuclear condensation and characteristic apoptotic bodies. (C) Myeloma cells freshly isolated from a patient and grown in the presence of 5 mM HMBA for 24 hr, exhibit apoptotic bodies.

Figure 3

DNA strand breaks induced by HMBA in ARP-1 Cells: Dotblot plots of DNA content (x axis) and DNA strand break labeling by BrdUTP (y axis). Cells in G₁ fall between 100 and 250 on the x axis, S-phase cells and G₂/M-phase cells lie between 250 and 600 on the x axis.

Figure 4

BCL-2 expression in ARP-1 cells. (A) Western blot analysis of BCL-2 protein level in ARP1 cells cultured without and with HMBA for 12, 24, and 48 hr. KG1a is a leukemia cell line overexpressing BCL-2, used as a positive control. (B) Myeloma colony from a patient positively stained with anti-BCL-2 mAb. (C) 24 hr after exposure to 5 mM HMBA, the cells no longer stain for BCL-2 protein and display apoptotic morphological features.