ABSTRACT
A number of polyamine analogs are currently used in various clinical trials as cancer treatment and it is important to investigate their effects not only on cancer cells but also on normal cells. Treatment with polyamine analogs depletes cells of polyamines and inhibits cell proliferation, but the analogs cannot take over the normal function of the natural polyamines in the cell. In this study, the normal-like breast epithelial cell line MCF-10A was treated with the polyamine analog N',N"-diethylnorspermine (DENSPM). The cells were then studied using a bromodeoxyuridine- DNA flow cytometry method as well as western blot. The ability of both normal-like and breast cancer cells to recover from DENSPM treatment was also studied. DENSPM treatment of MCF-10A cells resulted in a prolongation of the S and G2 +M phases, followed by a G1/S block. The p53/p21/RB1 pathway was involved in the G1/S block as shown by increased levels of p53 and p21 detected by western blot. Decreased levels of cyclin E1, cyclin A2, and cyclin B1 in DENSPM-treated cells can explain the prolongation of cell cycle phases that occurred before the G1/S block. We also show that MCF-10A cells rapidly recover from DENSPM-induced growth inhibition in contrast to four human breast cancer cell lines. The goal of cancer treatment is to cause minimal and reversible damage to normal cells, while cancer cells should be eliminated. Altogether, the data show that treatment with polyamine analogs spares normal cells, while negatively affecting the cancer cells.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle/drug effects , Spermine/analogs & derivatives , Blotting, Western , Breast Neoplasms/pathology , Bromodeoxyuridine , Cell Line , Cell Line, Tumor , Cyclin A/drug effects , Cyclin A/metabolism , Cyclin A2 , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin B1 , Cyclin E/drug effects , Cyclin E/metabolism , DNA , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Flow Cytometry , Humans , Oncogene Proteins/drug effects , Oncogene Proteins/metabolism , Spermine/pharmacologyABSTRACT
Polyamine analogues are presently undergoing clinical evaluation in the treatment of cancer. To better understand under what circumstances treatment with a polyamine analogue will yield beneficial results, we have investigated the effect of N,N-diethylnorspermine (DENSPM) on cell cycle kinetics of the human breast cancer cell lines SK-BR-3, MCF-7, HCC1937, and L56Br-C1. A bromodeoxyuridine-DNA flow cytometry method was used to evaluate the treatment with 10 micromol/l DENSPM on cell cycle kinetics. A correlation between polyamine pool size after DENSPM treatment and cell cycle kinetic effects was found. The most sensitive cell cycle phase was the S phase, followed by an effect on the G2+M phase and then the G1/S transition. The levels of a number of cell cycle regulatory proteins such as cyclin E1, cyclin A2, and cyclin B1 were lowered by DENSPM treatment, which may explain the effects on cell cycle kinetics. The two cell lines that were most sensitive to DENSPM treatment belong to the basal-like subtype of breast cancer and they were deficient with respect to p53, BRCA1, and RB1.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Polyamines/pharmacology , Spermine/analogs & derivatives , Blotting, Western , Breast Neoplasms , Bromodeoxyuridine/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cyclins/metabolism , Female , Flow Cytometry , Humans , Microscopy, Fluorescence , Spermine/pharmacologyABSTRACT
Polyamines are organic cations, which are considered essential for normal cell cycle progression. This view is based on results from numerous studies using a variety of enzyme inhibitors or polyamine analogues interfering with either the metabolism or the physiological functions of the polyamines. However, the presence of non-specific effects may be hard to rule out in such studies. In the present study, we have for the first time used a transgenic cell system to analyze the importance of polyamines in cell growth. We have earlier shown that expression of trypanosomal ODC in an ODC-deficient variant of CHO cells (C55.7) supported growth of these otherwise polyamine auxotrophic cells. However, one of the transgenic cell lines grew much slower than the others. As shown in the present study, the level of ODC activity was much lower in these cells, and that was reflected in a reduction of cellular polyamine levels. Analysis of cell cycle kinetics revealed that reduction of growth was correlated to prolongation of the G1, S, and G2+M phases in the cells. Providing exogenous putrescine to the cells resulted in a normalization of polyamine levels as well as cell cycle kinetics indicating a causal relationship.
