Abstract
Mutations in the clk-2 gene of the nematode Caenorhabditis elegans affect organismal features such as development, behavior, reproduction, and aging as well as cellular features such as the cell cycle, apoptosis, the DNA replication checkpoint, and telomere length. clk-2 encodes a novel protein (CLK-2) with a unique homologue in each of the sequenced eukaryotic genomes. We have studied the human homologue of CLK-2 (hCLK2) to determine whether it affects the same set of cellular features as CLK-2. We find that overexpression of hCLK2 decreases cell cycle length and that inhibition of hCLK2 expression arrests the cell cycle reversibly. Overexpression of hCLK2, however, renders the cell hypersensitive to apoptosis triggered by oxidative stress or DNA replication block and gradually increases telomere length. The evolutionary conservation of the pattern of cellular functions affected by CLK-2 suggests that the function of hCLK2 in humans might also affect the same organismal features as in worms, including life span. Surprisingly, we find that hCLK2 is present in all cellular compartments and exists as a membrane-associated as well as a soluble form.
Highlights
Identifying and studying the processes and the genes that are involved in determining the rate of aging is a challenging area of modern genetics
Overexpression of homologue of CLK-2 (hCLK2) Produces Hypersensitivity to Apoptosis Triggered by Oxidative Stress or DNA Replication Block—Prompted by the findings in the germ line of C. elegans, where clk-2 mutations affect the response to ionizing radiation and to DNA replication block induced by HU, we investigated the response of SK-HEP-1 cells overexpressing hCLK2 to 10 different agents capable of inducing apoptotic cell death as well as to HU and ␥-rays
The broad pleiotropy observed in clk-2 mutants suggests that, in worms, the function of clk-2 encodes a novel protein (CLK-2) links important cellular processes such as cell cycle control, apoptosis, and telomere length regulation
Summary
Cell Culture—All cells were grown in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (plus nonessential medium amino acids for HT-1080 and SK-HEP-1) at 37 °C in an atmosphere of 5% CO2 and 95% air. Construction of the Plasmid pLXSH-hclk and Establishment of a Stable Cell Line Overexpressing hCLK2—A cDNA clone hk02952 (insert size 4337 bp), containing the full-length hclk cDNA sequence, as well as parts of intronic sequences (1929 –2171, 2288 –2456, and 2812–3434) was obtained from Kazusa DNA Research Institute, Japan. Using this cDNA as a template, two fragments that exclude the intron sequences, ⌬hclk2-A (from bp 256 to 1929) and ⌬hclk2-B (from bp 1929 to 3434) were generated by PCR and cloned into a pcDNA3.1/V5/His/TOPO vector (Invitrogen) to produce pcDNA3.1-⌬hclk2-A and pcDNA3.1⌬hclk2-B. Cells were exposed to the siRNA treatment on day 1, and they were passaged 1:4 on day 4
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