p53-independent inhibition of proliferation and p21(WAF1/Cip1)-modulated induction of cell death by the antioxidants N-acetylcysteine and vitamin E.

Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, and of the cyclin-dependent kinase (CDK) inhibitor p21(Waf1/Cip1). The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21(Waf1/Cip1), p18(ink4c) or p16(ink4a), but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21(Waf1/Cip1) increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21(Waf1/Cip1) and basal levels of ROS are important determinants of outcome after antioxidant treatment.

Cloning and characterization of a cdc25 phosphatase from mouse lymphocytes.

Members of the cdc25 phosphatase family are proposed to function as important regulators of the eukaryotic cell cycle, particularly in the induction of mitotic events. A new cdc25 tyrosine phosphatase, cdc25M1, has been cloned from a mouse pre-B cell cDNA library and characterized. The cdc25M1 protein consists of 465 amino acids with a predicted relative molecular mass (M(r)) of 51,750. Over the highly conserved carboxyl terminal region, the amino acid sequence similarity to the human cdc25 C or Hs1 isoform is 89%, while the overall similarity is 67%. The phosphatase active site is located within residues 367-374. Tissue expression of the cdc25M1 was highest in mouse spleen and thymus by northern blot analysis. The cdc25M1 mRNA was detected in a number of cloned mouse lymphocyte cell lines including both CD8+ and CD4+ cells. cdc25M1 mRNA was shown to be cell cycle-regulated in T cells following interleukin-2 (IL-2)-stimulation. Accumulation of cdc25M1 mRNA occurred at 48 h after IL-2 stimulation, when lymphocytes were progressing from S phase to G2/M phase of the cell cycle. This pattern of expression is in contrast to that observed for other protein tyrosine phosphatases expressed in T lymphocytes including CD45, LRP, SHP, and PEP. The elevation in cdc25M1 mRNA level occurred concomittant to the appearance of the hyperphosphorylated form of p34cdc2 protein kinase. A purified, bacterial-expressed recombinant cdc25M1 phosphatase domain catalyzed the dephosphorylation of p-nitrophenol phosphate, as well as [32P-Tyr] and [32P-Ser/Thr]-containing substrates. Preincubation of p34cdc2 kinase with cdc25M1 activated its histone H1 kinase activity in vitro. These results suggest that cdc25M1 may be involved in regulating the proliferation of mouse T lymphocytes following cytokine stimulation, through its action on p34cdc2 kinase.