Topoisomerase I activity in irradiated Chinese hamster cells: relation to chromosomal aberrations and cell cycle progression

Topoisomerase (TOP) I plays a major role in the process of supercoiled DNA relaxation, thereby facilitating DNA replication and cell cycle progression. The expression and enzymatic activity of TOP I is positively correlated with tumor progression. Although the anticancer activity of (S)-10-Hydroxycamptothecin (HCPT), a TOP I specific inhibitor, has been reported in various cancers, the effect of HCPT on esophageal cancer is yet to be examined. In this study, we investigate the potential of HCPT to inhibit the growth of ESCC cells in vitro and verify its anti-tumor activity in vivo by using a patient-derived xenograft (PDX) tumor model in mice. Our study revealed the overexpression of TOP I in ESCC cells and treatment with HCPT inhibited TOP I enzymatic activity at 24 h and decreased expression at 48 h and 72 h. HCPT also induced DNA damage by increasing the expression of H2A.XS139. HCPT significantly decreased the proliferation and anchorage-independent growth of ESCC cells (KYSE410, KYSE510, KYSE30, and KYSE450). Mechanistically, HCPT inhibited the G2/M phase cell cycle transition, decreased the expression of cyclin B1, and elevated p21 expression. In addition, HCPT stimulated ESCC cells apoptosis, which was associated with elevated expression of cleaved PARP, cleaved caspase-3, cleaved caspase-7, Bax, Bim, and inhibition of Bcl-2 expression. HCPT dramatically suppressed PDX tumor growth and decreased the expression of Ki-67 and TOP I and increased the level of cleaved caspase-3 and H2A.XS139 expression. Taken together, our data suggested that HCPT inhibited ESCC growth, arrested cell cycle progression, and induced apoptosis both in vitro and in vivo via decreasing the expression and activity of TOP I enzyme.

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Insulin-Induced Cell Cycle Progression Is Impaired in Chinese Hamster Ovary Cells Overexpressing Insulin Receptor Substrate-3

Endocrinology ◽

10.1210/en.2004-0199 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5862-5874 ◽

Cited By ~ 10

Author(s):

Yasushi Kaburagi ◽

Ryo Yamashita ◽

Yuzuru Ito ◽

Hitoshi Okochi ◽

Ritsuko Yamamoto-Honda ◽

...

Keyword(s):

Cell Cycle ◽

Insulin Receptor ◽

Cyclin D1 ◽

Cell Cycle Progression ◽

Glycogen Synthase ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Glycogen Synthase Kinase 3 ◽

Cycle Progression ◽

Ovary Cells

Abstract To analyze the roles of insulin receptor substrate (IRS) proteins in insulin-stimulated cell cycle progression, we examined the functions of rat IRS-1 and IRS-3 in Chinese hamster ovary cells overexpressing the human insulin receptor. In this type of cell overexpressing IRS-1 or IRS-3, we showed that: 1) overexpression of IRS-3, but not IRS-1, suppressed the G1/S transition induced by insulin; 2) IRS-3 was more preferentially localized to the nucleus than IRS-1; 3) phosphorylation of glycogen synthase kinase 3 and MAPK/ERK was unaffected by IRS-3 overexpression, whereas that of protein kinase B was enhanced by either IRS; 4) overexpressed IRS-3 suppressed cyclin D1 expression in response to insulin; 5) among the signaling molecules regulating cyclin D1 expression, activation of the small G protein Ral was unchanged, whereas insulin-induced gene expression of c-myc, a critical component for growth control and cell cycle progression, was suppressed by overexpressed IRS-3; and 6) insulin-induced expression of p21, a cyclin-dependent kinase inhibitor, was decreased by overexpressed IRS-3. These findings imply that: 1) IRS-3 may play a unique role in mitogenesis by inhibiting insulin-stimulated cell cycle progression via a decrease in cyclin D1 and p21 expressions as well as suppression of c-myc mRNA induction in a manner independent of the activation of MAPK, protein kinase B, glycogen synthase kinase 3 and Ral; and 2) the interaction of IRS-3 with nuclear proteins may be involved in this process.

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SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

The Journal of Cell Biology ◽

10.1083/jcb.58.2.340 ◽

1973 ◽

Vol 58 (2) ◽

pp. 340-345 ◽

Cited By ~ 14

Author(s):

Kenneth D. Ley ◽

Marilyn M. Murphy

Keyword(s):

Cell Cycle ◽

Mitochondrial Dna ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Time Course ◽

Nuclear Dna ◽

Tritiated Thymidine ◽

Chinese Hamster ◽

Chinese Hamster Cells ◽

In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

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