Human GST5.8, Expressed in a Cell Cycle Specific Manner, May Offer a Method to Alter 4-Hydroxynonenal Concentrations of Philadelphia-Positive Chronic Myelogenous Leukemia Cells.

Chronic Myelogenous Leukemia studies in K562 cell line, obtained from the pleural effusion of a CML patient in blastic crisis, initially focused upon chromosomal translocations such as del(22) (q12) and t(15;17)(q21;q24). {{Lozzio,C.B. 1975}} In a 2001 Phase I Clinical Trial, STI-571, a tyrosine kinase inhibitor which treats the BCR/Abl fusion protein formed due to t(9;22)(q34;q11) found in the majority of CML patients effectively ceased proliferation of BCR-Abl expressing hematopoietic cells.{{Brian J. Druker, M.D., 2001}} However, recent studies demonstrate that Chronic Myelogenous Leukemia persists in patients exhibiting differing chromosomal abnormalities. {{Jamshid S Khorashad, MD, 2008}} Therefore, we examined methods for inducing differentiation in K562 cells to pursue the senescence of leukemic blasts beyond proliferation in heterogeneic CML populations. Using 20 uM 4-hydroxynonenal, a lipid peroxidation product, Cheng successfully produced differentiated K562 cells. {{Cheng,J.Z. 1999}} Our studies confirm this; however 20 um-HNE-treated K562 cells also aggregate. Here, we performed titration studies examining extremely low concentrations of 4-HNE and its correlation with K562 morphology to investigate if low-HNE-containing cells remained premature blasts. Barrera also demonstrated inhibition of cell proliferation, as well as the downregulation of c-myc expression in K562 cells, by HNE in a dose-dependent manner. {{Barrera,G. 2004}} However, he reported that these effects were transient and disappeared within 6–8 hours following HNE administration. Because levels of aldehydes, such as 4-HNE, formed by lipid peroxidation are regulated through conjugation to glutathione, we investigated glutathione-S-transferase activity in K562 cells, which has been shown to exhibit extremely high specificity for 4-hydroxynonenal and is known to reduce 4-HNE concentrations in tissue cells. {{Cheng,J.Z. 1999}}

Human GST5.8 exhibits the highest specific activity of the glutathione-S-transferases toward lipid peroxidation product, 4-HNE as shown in tissue. {{Singhal,S.S. 1999}} So, we observed if higher concentrations of the enzymatic activity occurred at specific points in the K562 cell cycle, affecting HNE concentrations and contributing to cell cycle phases such as G0/G1 or S phase.

We examined human GST5.8 levels in K562 cells following synchronized release from arrest with sodium butyrate. In addition, we performed morphological analysis of HNE effects on K562 cells via Giemsa stained negative controls versus titrations of HNE-treated K562 cells (at 0.5-20 uM) for 2 hours. Also, Western Blots and FACS analyses identifying time points of highest human GST5.8 expression were produced.

Densitometric analysis of hGST5.8 expression by time point revealed that human GST5.8 is present in K562 cells and that the enzymatic concentration peaks at 0 hr to 30 minutes following release from arrest or the G0/G1 time point. Our results confirmed that 20 um HNE causes differentiation, aggregation, and apoptosis in K562 cells. We also showed 0.5 uM concentration produces differentiation but very minimal aggregation.

Because human GST5.8 peaks during the G0/G1 time point, causing a reduction of the 4-hydroxynonenal concentration; it is reasonable to conclude that hGST5.8 may rise during G0/G1 in order to lower HNE concentrations as a dedifferentiation mechanism in normal cell cycle events. Here, we have demonstrated that human GST5.8 is expressed in a cell-cycle specific manner. Future studies involve transfection of hGST5.8 antisense into K562 to learn if consistent induction of differentiation is possible. Taken together, these studies indicate that hGST5.8 modulation may hold promise as a method to control 4-hydroxynonenal levels and thus differentiation in Ph(+) Chronic Myelogenous Leukemia cells.

No relevant conflicts of interest to declare.