Inhibition of c-Myc Oncoprotein Limits the Growth of Human Melanoma Cells by Inducing Cellular Crisis

Abstract

Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated ␤-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, progressive telomere shortening, glutathione (GSH), depletion and, increased production of reactive oxygen species. Treatment of control cells with L-buthionine sulfoximine decreases GSH to levels of cMyc low expressing cells, but it does not modify the growth kinetic of the cells. Surprisingly, when GSH is increased in the c-Myc low expressing cells by treatment with N-acetyl-L-cysteine, cells escape crisis. To test the hypothesis that both oxidative stress and telomerase dysfunction are involved in the c-Myc-dependent crisis, we directly inhibited telomerase function and glutathione levels. Inactivation of telomerase, by expression of a catalytically inactive, dominant negative form of reverse transcriptase, reduces cellular lifespan by inducing telomere shortening. Treatment of cells with L-buthionine sulfoximine decreases GSH content and accelerates cell crisis. Analysis of telomere status demonstrated that oxidative stress affects c-Myc-induced crisis by increasing telomere dysfunction. Our results demonstrate that inhibition of c-Myc oncoprotein induces cellular crisis through cooperation between telomerase dysfunction and oxidative stress.The proliferative potential of normal cells in culture is limited to a finite number of population doublings, a phenomenon known as cellular senescence or Hayflick limit (1) that is characterized by a large, flat morphology, telomere shortening, a high frequency of nuclear abnormality and induction of ␤-galactosidase activity (2, 3). Continued cell proliferation beyond the Hayflick limit and further telomere erosion culminates in a period of massive cell death termed crisis (3). The limited capacity of human cells to replicate is an important tumorsuppressive mechanism (4), indicating that cancer cells must overcome this obstacle and achieve proliferation immortality before they can form malignant neoplasms. Direct experimental evidence implicates telomere erosion as a primary cause of cellular arrest (5, 6). Most cells with indefinite proliferative ability maintain telomeres through the expression of the catalytic subunit of the enzyme, the human telomerase reverse transcriptase (hTERT) 1 (7,8), and the introduction of telomerase into normal human cells provides telomere maintenance, prevention of senescence or crisis, and extension of life span (5, 6). In the opposite type of experiment, inhibition of telomerase in established tumor cell lines induces telomere shortening, leading to chromosome end-to-end fusions, cell death, and eventually arrest of culture growth (9 -11). Recent evidence suggests that although telomere length is an important trigger for the onset of senescence, ...

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Anti-tumor Activity of the Ginsenoside Rk1 in Human Hepatocellular Carcinoma Cells through Inhibition of Telomerase Activity and Induction of Apoptosis

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