Increased levels of multiple forms of dihydrofolate reductase in peripheral blood leucocytes of cancer patients receiving haematopoietic colony-stimulating factors: interim analysis.

M Perwaiz Iqbal,Tariq Siddiqui,Fakhra Sultana,Ikram A Burney,Naseema Mehboobali

doi:10.1038/emm.2000.15

M Perwaiz Iqbal, Tariq Siddiqui + Show 3 more

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https://doi.org/10.1038/emm.2000.15

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Journal: Experimental and Molecular Medicine	Publication Date: Jun 1, 2000
Citations: 15	License type: cc-by

Abstract

The precise mechanism whereby granulocytes proliferate when haematopoietic colony stimulating factors (CSFs) are used in neutropenic cancer patients is poorly understood. The purpose of this study was to investigate whether these cytokines bring about leucocyte proliferation by increasing the levels of multiple forms of dihydrofolate reductase (DHFR). Blood samples were collected from 36 cancer patients (25 males and 11 females) with chemotherapy-induced neutropenia. One sample of blood from each patient was obtained before therapy either with CSF, such as granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) or with placebo, and another one at the time of resolution of neutropenia. Peripheral blood leucocytes in these blood samples were counted, separated and lysed. From lysates, cytoplasmic samples were prepared and analyzed for active DHFR by a methotrexate-binding assay and for total immunoreactive DHFR by an enzyme linked immunosorbent assay. The increase in total leucocyte count (TLC) was most prominent (P < 0.005) in the CSF group and less so (P < 0.05) in the placebo group. The mean +/- SD concentration values of active DHFR before and after stimulation with GM-CSF found were to be 0.34 +/- 0.4 ng/mg protein and 0.99 +/- 0.82 ng/mg protein, respectively, and in the group treated with G-CSF, 0.24 +/- 0.32 ng/mg protein and 1.18 +/- 2.4 ng/mg protein, respectively. This increase in active DHFR after stimulation with CSF was statistically significant (P < 0.05). Similarly, concentration values of immunoreactive but nonfunctional form of DHFR (IRE) were 110 +/- 97 ng/mg protein and 605 +/- 475 ng/mg protein before and after stimulation with GM-CSF, and 115 +/- 165 ng/mg protein and 1,054 +/- 1,095 ng/ mg protein before and after stimulation with G-CSF. This increase in concentration of IRE after stimulation with GM-CSF or G-CSF was statistically significant (P < 0.005). In the control group, there was an increase in the concentration of both active DHFR and IRE after treatment with placebo. However, this was not statistically significant. Resolution of neutropenia was quicker in the groups treated with CSF compared to the control group. Results of this study indicate that colony stimulating factors (G-CSF and GM-CSF) induce white cell proliferation by increasing the levels of multiple forms of DHFR.

Highlights

Haematopoietic colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are increasingly used to overcome the cytopenic side effects of chemotherapy (Vose et al, 1995; Raval et al, 1998)
Since dihydrofolate reductase (DHFR) has a key role in cell proliferation, it could be one of the enzymes influenced by CSFs
This has been identified in human leukaemia cells and in leucocytes of cancer patients and in normal colon as well as in colonic tumor tissues (Iqbal et al, 1992). The concentration of this immunoreactive nonfunctional form of enzyme (IRE) in human colon cytoplasm samples was nearly one tenth those of leukaemia cells suggesting that cells with greater proliferative potential may have a higher concentration of this protein

Summary

Introduction

Haematopoietic colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are increasingly used to overcome the cytopenic side effects of chemotherapy (Vose et al, 1995; Raval et al, 1998). Immunologically distinct and functionally inactive forms of this enzyme have been reported to be present in human leukemia cells and leucocytes of cancer patients (Iqbal and Rothenberg, 1981; Rothenberg and Iqbal, 1982; Iqbal et al, 1992; Iqbal et al, 1997). The concentration of this immunoreactive nonfunctional form of DHFR has been found to be greatly increased in cells during their log growth compared to the stationary growth phase (Rothenberg and Iqbal, 1983) suggesting that this form of enzyme may be having a role in cell proliferation. Studies in vitro have demonstrated that resting cells can be stimulated to re-enter the cell cycle through the addition of a factor in serum, which causes increased expression of DHFR gene (Johnson et al, 1978)

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