Prolonged early G1 arrest by selective CDK4/CDK6 inhibition sensitizes myeloma cells to cytotoxic killing through cell cycle-coupled loss of IRF4

Dysregulation of cyclin-dependent kinase 4 (CDK4) and CDK6 by gain of function or loss of inhibition is common in human cancer, including multiple myeloma, but success in targeting CDK with broad-spectrum inhibitors has been modest. By selective and reversible inhibition of CDK4/CDK6, we have developed a strategy to both inhibit proliferation and enhance cytotoxic killing of cancer cells. We show that induction of prolonged early-G(1) arrest (pG1) by CDK4/CDK6 inhibition halts gene expression in early-G(1) and prevents expression of genes programmed for other cell-cycle phases. Removal of the early-G(1) block leads to S-phase synchronization (pG1-S) but fails to completely restore scheduled gene expression. Consequently, the IRF4 protein required to protect myeloma cells from apoptosis is markedly reduced in pG1 and further in pG1-S in response to cytotoxic agents, such as the proteasome inhibitor bortezomib. The coordinated loss of IRF4 and gain of Bim sensitize myeloma tumor cells to bortezomib-induced apoptosis in pG1 in the absence of Noxa and more profoundly in pG1-S in cooperation with Noxa in vitro. Induction of pG1 and pG1-S by reversible CDK4/CDK6 inhibition further augments tumor-specific bortezomib killing in myeloma xenografts. Reversible inhibition of CDK4/CDK6 in sequential combination therapy thus represents a novel mechanism-based cancer therapy.     

An Ultrasensitive Radioligand Assay for IgC Using the Protein A on Staphylococcus aureau Bacteria

The purpose of this study was to develop a simple and sensitive assay to measure IgG. Human IgG was radiolabelled with ¹²⁵Iodine and 7.5 ng was incubated with heat-killed Staphylococcus aureus bacteria (Cowan 1 strain). To replicate sets of tubes, increasing amounts of a standard IgG preparation were added. The samples were incubated at room temperature for two hours and separated by centrifugation. Using this assay it was found that the IgG concentration could readily be determined in one nanoliter or less of human serum. There was no significant cross-reactivity with IgA, IgE and IgM or the F(ab')₂ fragment of IgG. Serial dilutions of normal human or SLE sera, rabbit or guinea pig sera, the Fc fragment of human IgG and a mouse monoclonal anti-human DNA antibody parallelled the dose response curve obtained with standard human IgG. The method correlated well (r=0.89) with a routinely used nephelometric method. The mean (±SD) IgG concentration in 20 normal subjects measured by this assay was 10 ± 3.6 g/L.