Besides their particular morphology, the mature myeloid cells can be distinguished with immunophenotypic changes and with a limited proliferation capacity. The proliferation arrest is mainly observed in cell cycle checkpoints. Several factors known as cyclin dependent kinase inhibitors (CDKIs) function as negative regulators of the cell cycle. There are two families of CDKIs: INK4 inhibitors and CIP/KIP inhibitors. Of the members of CIP/KIP family p21^Waf1/Cip1, p27^Kip1, p57^Kip2 can inhibit cyclin dependent kinases in any phase of the cell cycle, especially during G1/S transition[6,7].

Phorbol esters (e.g. phorbol 12-myristate 13-acetate (PMA), 12-O-tetradecanoyl phorbol-13-acetate (TPA)) are widely used as potent agents for HL-60 differentiation studies. These agents work through protein kinase C (PKC) molecules and activate transcription factors such as AP-1, NF-κB and mitogen-activated protein kinases (MAPKs)[1,8]. PMA may induce proliferation in some cell types, but it may lead to growth arrest in others such as HL-60 via blocking the cell cycle progression in G1/S checkpoint utilizing p21^Waf1/Cip1 and p27^Kip1 molecules[9].

It is well known that steroid hormones such as progesterone, β-estradiol and hydrocortisone play essential roles in differentiation and proliferation of cancer cells. In breast cancer cell lines, early effect of progestagens is acceleration of G1-S transition; however, following the completion of cell cycle, the cells are arrested at G1 phase via increased expression of p21^Waf1/Cip1. In addition, prolonged treatment with progesterone can result in overexpression of and leads to differentiation and/or apoptosis in other cell types[10]. On the other hand, estradiol regulates cell cycle progression and impairs the p21^Waf1/Cip1- and p27^Kip1-associated cell cycle arrest. Therefore, estrogen can reduce the amount of CDKI associated CDKs and promote cell cycle progression[11]. Hydrocortisone, acting through glucocorticoid receptors, can interfere with cell proliferation in G1 and S phases by increasing the expression of CDKIs[12].

The aim of the current study was to investigate the effect of the steroid agents, hydrocortisone, progesterone and β-estradiol, on the expression of cell cycle regulatory molecules p21^Waf1/Cip1, p27^Kip1 and p57^Kip2 in acute myeloid leukemia cell line HL-60 in accordance with morphological features.

Assessment of cell morphology and viability
Cell viability was determined according to propidium iodide (PI) exclusion method, and the viable cells were gated for morphological analysis by flow cytometry (EPICS XL-MCL, Beckman Coulter, Fullerton, CA, USA). The size and granularity of the cells were determined with forward scatter (FS) and side scatter (SS) parameters, respectively. The morphology was also confirmed by cytochemical staining using the standard Giemsa procedure.

Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated with QIAamp^® RNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. cDNA was synthesized from 2 μg of RNA, using oligo(dT) primers and RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Lithuania) according to manufacturer’s instructions. Previously reported primer oligonucleotide sequences were used to amplify p21^Waf1/Cip1, p27^Kip1 and p57^Kip2 genes[13]. Bactin gene was amplified as house keeping gene. PCR conditions are shown in Table-1. The cycling conditions for β-actin and p21^waf/cip were 30"’ at 94ºC; 30"’ at 58ºC; 45" at 72ºC; for p27^Kip1 were 30" at 94ºC; 30" at 55ºC; 45" at 72ºC, and for p57^kip2 were 30" at 94ºC; 30" at 62ºC; 45" at 72ºC. RNA samples isolated from peripheral blood mononuclear cells (PBMCs) were used for positive control amplifications.

Table 1: Final concentrations of the reagents used for p21^waf/cip, p27^kip1 and p57^kip2 RT-PCR

Preliminary reactions had been set to determine PCR cycle number which meets logarithmic amplification phase for each primer pair. Three sequential independent PCR amplifications were performed for each gene and reactions were terminated at the predetermined cycle. Products were resolved on 1% agarose gels and visualized with ethidium bromide staining under UV light.

Fig 1(A,B): (A): The morphological features of HL-60 cells treated with hydrocortisone (HC; 4.0 ug/ml), progesterone (PG; 0.5 ug/ml), phorbol 12-myristate 13-acetate (PMA; 10 nM), β-estradiol (β-E; 50 ng/ml) was analysed by flow cytometry. Representative results are shown. (B): Size (FS) and granularity (SS) values of three independent experiments are displayed in percentages

The expression of p21^Waf1/Cip1, p27^Kip1 and p57Kip2 genes in steroid-treated HL-60 cells
Cellular differentiation is usually accompanied with changes in cell cycle dynamics especially regulated at G1/S transition check point[10]. Therefore, following the steroid agent treatments, we analyzed the expression of p21^Waf1/Cip1, p27^Kip1 and p57^Kip2 genes (Figure 2).

Fig 2: The expression of p21^Waf1/Cip1, p27^Kip1 and p57^Kip2 genes in steroid-treated HL-60 cells. cDNA synthesized from PBMCs were used as positive control for p57^Kip2 RT-PCR M: 50 bp DNA marker; HC: hydrocortisone; PG: progesterone; PMA: phorbol 12-myristate 13-acetate; β-E: β-estradiol)

In the HL-60 cells which are incubated without steroid agents, p21^Waf1/Cip1 expression was lower than p27^Kip1 whereas no p57^Kip2 expression was observed. Highest expression of p21^Waf1/Cip1 was detected in progesteronetreated HL-60 cells; on the other hand, p27^Kip1 was moderately decreased in these cells compared to the control group. All agents caused a decrease in p27Kip1 expression with β-estradiol causing the highest decrease. Furthermore, hydrocortisone had the mildest effect on HL-60 cells in terms of p21^Waf1/Cip1 and p27^Kip1 expression when compared to control cells. As the agent resulting in morphological differentiation, PMA treatment increased the p21^Waf1/Cip1 expression while decreasing the p27^Kip1 expression. Collectively, a reciprocal relationship was observed between expression levels of p21^Waf1/Cip1 and p27^Kip1 as a decline in one molecule’s expression was found to be accompanied by an increase in the other, or vice versa. With the exception of the β-estradioltreated cells, the expression of both genes was relatively decreased. Moreover, no p57^Kip2 expression was detected in none of the experimental groups whereas it could be amplified in PBMCs.

Steroids are important regulators in normal development of certain tissues such as mammary, endometrial, alveolar and intestinal epithelia[15]. Moreover, progesterone and estrogens are implicated in oncogenesis[16]. Glucocorticoids are clinically effective for the treatment of prostate cancer[17]. It is well established that the steroids influence the cellular differentiation and proliferation by modulating the activities of cyclins and CDKIs[18].

Cyclins, CDKs and their negative regulators, CDKIs, are responsible for regulating cell-cycle checkpoints[6]. The CDKIs analyzed in our study are involved in G1/S checkpoint, the first control step after the cells are induced to proliferate and not to differentiate. HL-60 myeloid leukemia cells have a high proliferation index, however they may be subject to some degree of spontaneous differentiation[19]. In our study we used the phorbol ester, PMA, a potent agent differentiating the HL-60 cells through protein kinase C pathway[1,20]. The expression of p21^Waf1/Cip1 and p27^Kip1 genes are implicated in the PMAinduced cell cycle block[9]. Accordingly, in our study, a prominent increase in p21^Waf1/Cip1 expression was observed in PMA-treated cells, on the other hand, p27^Kip1 was not augmented at the time of analysis.

Estradiol can modulate the cell cycle indirectly through intracellular signalling pathways and directly as a transcription factor. Estrogen favors cell cycle progression, decreases the formation of p21^Waf1/Cip1- and p27^Kip1-cyclinE/Cdk2 complex[11]. The reducing effect of b-estradiol on CDKI, especially p21^Waf1/Cip1, expression in HL-60 myeloid leukemia cells may also indicate an increase in proliferation. Accordingly, the acute myeloid leukemia cases in which estrogen receptors are found to be methylated have a relatively better prognosis[21].

In breast cancer cell lines, progesterone is initially proliferative, however, following the completion of the cell cycle the increased expression of p21^Waf1/Cip1 leads to an arrest in G1/S transition[10]. Besides, prolonged exposure to progesterone cause the overexpression of p21^Waf1/Cip1 resulting in differentiation and apoptosis of the cells other than breast cancer. For the HL-60 cells treated with progesterone, a prominent increase in p21^Waf1/Cip1 expression was determined. On the other hand, no obvious change in morphology and viability was noted at 96 hours of incubation.

Glucocorticoid receptors are located intracellularly where they bind hydrocortisone in a cell cycle-dependent way. Glucocorticoids are reported to increase the expression of CDKIs, such as p21^Waf1/Cip1 and p27^Kip1, favoring cell cycle arrest in G1 or S phase[12]. In response to hydrocortisone, the expression of p27^Kip1 was higher than p21^Waf1/Cip1 in HL-60. Nonetheless, although a slight increase was seen in p21^Waf1/Cip1, the expression pattern was not far from the control cells.

p57^Kip2 gene is not deleted in HL-60 cell line but it is epigenetically inactivated by DNA methylation[7]. The absence of the CDKI p57^Kip2 may increase the importance of p21^Waf1/Cip1 and p27^Kip1 in cell cycle regulation. Although deletions have been demonstrated in HL-60 cell line, in genes encoding tumor suppressor protein p53, evidences show that growth factors and differentiation-inducing agents may provide p53-independent induction of CDKIs (e.g. p21^Waf1/Cip1) as an immediate gene response[1,22].

The overall data implied that G1/S transition in HL- 60 myeloid leukemia cells may be primarily regulated through p21^Waf1/Cip1 and with a lesser extent through p27^Kip1 expression upon incubation with steroids. In a previous study performed by our group, a similar phenomenon was observed in HL-60 cells grown on fibronectin matrix[19]. Although p21^Waf1/Cip1 and p27^Kip1 demonstrate redundant effects at the G1/S transition, the reciprocal but alternate change in the levels of expression may indicate their differential regulation upon certain stimuli.

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