Comparative detection and quantitation of human CDK inhibitor mRNA expression of p15^INK4B, p16^INK4A, p16β, p18^INK4C, p19^INK4D, p21^WAF1, p27^KIP1 and p57^KIP2 by RT‐PCR using a polycompetitive internal standard

Abstract

For comparative and quantitative analysis of human cyclin‐dependent kinase inhibitor gene expression (CKI; p15INK4B, p16INK4A, p16β, p18INK4C, p19INK4D, p21WAF1, p27KIP1 and p57KIP2) we set up an RT‐PCR assay with a construct termed pCKIquant producing polycompetitive RNA as an internal standard. We demonstrated the reproducibility, accuracy and high sensitivity of the assay in the in vitro model of myeloid leukaemic HL‐60 cells. We also showed that the pCKIquant CKI assay is an excellent tool for the assessment of CKI mRNA expression in clinical samples, e.g. single cryostat sections of lymphoma biopsies.

The cyclin‐dependent kinase inhibitors p18INK4c and p19INK4d are highly expressed in CD34+ progenitor and acute myeloid leukaemic cells but not in normal differentiated myeloid cells

“…This raises the hypothesis that a subset of CKIs is important in keeping haemopoietic stem cells in a resting state and thus preventing differentiation. This is supported by our previous ®nding that only these two CKIs are down-regulated during PMA-induced differentiation of HL60 cells (Schwaller et al, 1997a (Hirai et al, 1995). Mice lacking p18…”

“…Our RT-PCR assay is based on a multispeci®c CKI competitor (pCKIquant) described previously (Schwaller et al, 1997a). Brie¯y, the pCKIquant competitor was photometrically quantitated and diluted from 10 À5 to 10 À10 mg/ml.…”

“…Thus, disturbances of gene regulation at the transcriptional level during differentiation may be more important than genomic alterations. Previously we found exclusive down-regulation of p18 INK4c and p19 INK4d in HL60 leukaemic cells induced along the monocytemacrophage differentiation (Schwaller et al, 1997a present study we were mainly interested in investigating the expression pattern of these two CKIs in primary cells from AML patients.…”

“…in HL60 leukaemic cells induced along the monocytemacrophage differentiation (Schwaller et al, 1997a present study we were mainly interested in investigating the expression pattern of these two CKIs in primary cells from AML patients.…”

Aberrant FHIT mRNA transcripts are present in malignant and normal haematopoiesis, but absence of FHIT protein is restricted to leukaemia

“…No dierences in the band patterns of ampli®cation products were noted. RNA integrity was veri®ed by RT ± PCR ampli®cation of b-actin RNA (Schwaller et al, 1997). All PCR products were separated on 1.5% agarose gels and stained with ethidium bromide.…”