PCR and immunocytochemistry detection of p27[kip1] in chronic lymphocytic leukemia

ABSTRACT

Chronic B-cell lymphocytic leukemia [B-CLL] is a clonal expansion of B-cells with low proliferative activity in which the cells are arrested in G[0]/G[1] phase of cell cycle. p27[KIP1] is one of the KIP/CIP family of cyclin-dependent kinase inhibitors [CKIs] which inhibit all cyclin dependent kinases by direct binding to cdk complexes. It is highly expressed when cells are arrested in G[0]/G[1]and its expression declines as cells progress towards S phase. In B-CLL the non-physiological increase in p27[KIP1] appears to be of clinical relevance since high protein levels correlate with poorer survival of patients. To study the expression of p27[KIP1] in B-CLL patients and correlate these results with the clinical and laboratory data of patients. p27[KIP1] expression was determined at the mRNA level by semi-quantitative reverse transcriptase polymerase chain reaction [RT-PCR] and at the protein level by immunocytochemistry in 35 patients with de novo B-CLL and 30 healthy age- and sex-matched control subjects. p27[KIP1] mRNA levels by RT-PCR was significantly higher among CLL patients compared to the control subjects [p<0.001] and was significantly higher among group II CLL patients [lymphocyte count >30 x 10[3]/L] compared to group I CLL patients [lymphocyte count protein expression of p27[KIP1] by immunocytochemistry was significantly higher among the control subjects compared to CLL patients [p<0.001] and was significantly higher among group II CLL patients compared to group I. There was a significant positive correlation between p27[KIP1] mRNA levels and both total leucocytic count and absolute lymphocytic count [p<0.001 and <0.05, respectively]. As a prognostic marker, there was no correlation between p27[KIP1] mRNA levels and neither LDH nor other immunophenotypic markers as FMC[7], CD[23]. Increasing levels of p27[KIP1] RT-PCR were positively correlated with increasing frequency of nuclear positivity by immunocytochemical staining [p<0.001]. p27[KIP1] expression is more accurately measured at the transcription level by RT-PCR based technique in measuring its protein level using the immunocytochemistry. Also more prospective studies investigating p27[KIP1] may indeed provide important information on potential targets for therapeutic strategies