Semester

Spring

Date of Graduation

2002

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Jeannine S. Strobl.

Abstract

Previously we showed that quinidine arrested MCF-7 cells in G1 phase of the cell cycle and led to a G1 to G0 transition followed by apoptotic cell death (Wang et al., 1998). The present experiments demonstrate that MCF-7, MCF-7ras, T47D, MDA-MB-231, and MDA-MB-435 cells transiently differentiate before undergoing apoptosis in response to quinidine. The cells accumulated lipid droplets and the cytokeratin 18 cytoskeleton was reorganized. Hyperacetylated histone H4 appeared within 2 h of the addition of quinidine to the medium, and levels were maximal by 24 h. Quinidine treated MCF-7 cells showed elevated p21/WAF1, hypophosphorylation and suppression of retinoblastoma protein, and down-regulation of cyclin D1. Quinidine did not show evidence for direct inhibition of histone deacetylase enzymatic activity in vitro. HDAC-1 was undetectable in MCF-7 cells 30 min after addition of quinidine to the growth medium. The proteasome inhibitors, MG-132 and lactacystin completely protected HDAC-1 from the action of quinidine. These data demonstrate that quinidine is a breast tumor cell differentiating agent that causes the loss of HDAC-1 via a proteasomal sensitive mechanism. Moreover, to determine whether effects of quinidine on cell differentiation and apoptosis is generalizable to quinoline antimalarials, we tested typical quinoline antimalarial drugs for their ability to regulate MCF-7 cell differentiation, hyperacetylation histone H4 and apoptosis. MTS IC50 of amodiaquin, chloroquine, primaquine, quinidine as well as quinine induced hyperacetylation of histone H4 without inhibiting HDAC activity. These results indicate that an alkylamino-substituted quinoline ring might be important for acetylation of histone H4. Accumulation of lipid droplets was observed in cells treated with chloroquine, primaquine, quinidine and quinine. Only MTS IC50 of chloroquine and quinidine increased p21(WAF1) protein levels and apoptosis. The data imply that p21(WAF1) might play a determining role in apoptosis in MCF-7 cells treated with antimalarials. Chloroquine was most active in stimulating MCF-7 apoptosis, and quinine was most active in promoting MCF-7 cell differentiation. We conclude that distinct mechanisms are responsible for breast tumor cell differentiation and activation of apoptosis by quinoline antimalarials. Alkylamino-substituted quinoline ring compounds represented by quinidine, quinine and chloroquine will be useful model compounds in the search for more active breast tumor differentiating agents.

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