Cell cycle regulating kinase Cdk4 as a potential target for tumour visualisation in vivo

Aim
Loss of growth control in tumour cells is a result of alterations in regulatory pathways of the cell cycle, in particular, the cyclin-dependent kinase (Cdk)/retinoblastoma (pRb)/E2F cascade. Inhibition of this G1/S transition regulating pathway using, e.g., selective Cdk4 inhibitors provides a promising approach for cancer treatment.
We hypothesise that potent Cdk4 inhibitors are suitable for imaging of cell proliferation processes and tumour visualisation by PET in vivo. Therefore, biological, biochemical, and radiopharmacological characteristics of a selected Cdk4 inhibitor (CKIA, 8-Cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl-phenylamino)-8H-pyrido[2,3-d]pyrimidin-7-one) was examined.

Materials & Methods
Cell growth and cell cycle studies were performed in human tumour cell lines (HT-29, FaDu, THP-1). Cdk4 specific pRb phosphorylation at Ser780 was analysed in cell lysates by western blotting. mRNA expression of the pRb affected genes E2F-1 and PCNA was measured with quantitative RT-PCR. Further, cell uptake studies were performed using iodine-124 radiolabelled CKIA ([124I]CKIA).

Results
Tumour cell growth studies indicate a significantly reduced cell proliferation in all cell lines after 48 h of treatment with 0.1 (< 50%) and 1 µM (< 15%) CKIA. This effect is confirmed by cell cycle studies. Already 24 h after incubation with CKIA the percentage of tumour cells in G1 phase showed a concentration dependent increment up to 90%. pRb phosphorylation is decreased three- to tenfold after 24 h of treatment with 0.1 and 1 µM CKIA in all cell lines. Further, a downregulation of E2F-1 and PCNA mRNA expression could be demonstrated.
Preliminary experiments with [124I]CKIA indicate an adequate stability of this compound in various buffers (pH 4.2, 7.4, 9.0), cell culture media, and rodent plasma samples. In vitro cell uptake studies in adherent tumour cells using [124I]CKIA showed a substantial uptake in HT-29 (1,649±117 %ID/mg protein) and FaDu cells (1,033±84 %ID/mg protein) after 2 h at 37°C.

Conclusion
Our data demonstrate a definite and specific inhibition of tumour cell proliferation when incubating with CKIA due to an arrest of tumour cells in G1 phase. The repression of G1 progression is achieved by CKIA induced inhibition of the Cdk4/pRb/E2F pathway. The stability of [124I]CKIA in physiological buffers and plasma, and the cell uptake observed in human tumour cells are a promising prerequisite for in vivo biodistribution and imaging studies to further support our hypothesis that radiolabelled CKIA and corresponding compounds are suitable tracers for visualisation of tumours using PET.