Cell cycle kinase inhibitor expression and hypoxia-induced cell cycle arrest in human cancer cell lines

• Published in: Carcinogenesis (New York) Vol. 25; no. 12; pp. 2325 - 2335
• Main Authors: Box, Adrian Harold, Demetrick, Douglas James
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01-12-2004; Oxford Publishing Limited (England)
• Subjects: Apoptosis; Biological and medical sciences; Carcinogenesis, carcinogens and anticarcinogens; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - metabolism; CDK; CDKI; cell cycle kinase inhibitors; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Hypoxia; cyclin-dependent kinase; Cyclin-Dependent Kinase Inhibitor p16 - genetics; Cyclin-Dependent Kinase Inhibitor p16 - metabolism; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Fibroblasts - metabolism; G1 Phase; Glycogen Synthase Kinase 3 - genetics; Glycogen Synthase Kinase 3 - metabolism; Glycogen Synthase Kinase 3 beta; HeLa Cells; HIF-1α; HMEC; human mammary epithelial cells; Humans; hypoxia inducible factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; Medical sciences; p15; p15INK4a; p16; p16INK4a; p18; p18INK4a; p19; p19INK4a; p21; p21WAF1; p27; p27Kip1; p57; p57Kip2; Phosphorylation; pRb; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; retinoblastoma protein; Ribonuclease Protection assay; RNA, Messenger - genetics; RNA, Messenger - metabolism; RPA; S Phase; Transcription Factors - genetics; Transcription Factors - metabolism; Tumor Cells, Cultured; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Tumors; tumour suppressor; Human; Oxygen; Enzyme; Transferases; Malignant tumor; In vitro; Cell line; Kinase; Cell cycle; Established cell line; Hypoxia; Inhibitor; Tumor cell
• DOI: 10.1093/carcin/bgh274
• EISSN: 1460-2180
• ISSN: 0143-3334; 1460-2180

Flow cytometric analysis of fibroblasts, normal breast epithelial cells and breast or other cancer cell lines identified variation in the abilities of cell lines to undergo cell cycle arrest as a response to hypoxia. Human mammary epithelial cells (HMEC), normal fibroblasts (Hs68 and WI38), HeLa cervical carcinoma and HTB-30 breast carcinoma cells arrest in G1/S in response to severe hypoxia. Hep3B hepatocellular carcinoma cells did not exhibit orderly G1/S arrest in response to severe hypoxia. We found a general decrease in p16INK4a (p16) mRNA levels, with an associated decrease in p16 protein levels in both normal cells and in cancer cells, regardless of their cell cycle response to hypoxia. p27 protein levels did not correlate with the cell line's ability to enter a hypoxic G1/S arrest. Furthermore, cell lines that underwent G1/S arrest showed decreased expression of hypoxia inducible factor 1 (HIF-1α) and at least one member of INK4 or Sdi cell cycle kinase inhibitors families after 12–24 h of hypoxia. Conversely, Hep3B, which did not exhibit orderly hypoxia-associated G1/S arrest, also did not show decreased HIF-1α, INK4 or Sdi protein levels in hypoxia. Furthermore, Hep3B showed constitutive activating phosphorylation of Akt and inhibitory phosphorylation of GSK3β, which was the opposite pattern to that exhibited by the cell lines showing the G1/S arrest phenotype. Inhibition of GSK3β by lithium chloride treatment of HeLa cells converted the HIF-1α, p16 and p27 loss to levels unchanged by hypoxic exposure. Our results suggest that regulation of the cell cycle during hypoxia in either normal or cancer cells is not simply due to up-regulation of cell cycle kinase inhibitors. Furthermore, decreased protein expression of HIF-1α, p16 and p27 was associated with both a hypoxia-induced G1/S arrest phenotype and increased GSK3β activity.