Monoclonal antibody probes for p21WAF1/CIP1 and the INK4 family of cyclin-dependent kinase inhibitors.

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

Dissecting functions of the retinoblastoma tumor suppressor and the related pocket proteins by integrating genetic, cell biology, and electrophoretic techniques.

The members of the 'pocket protein' family, comprising the retinoblastoma tumor suppressor (pRB) and its relatives, p107 and p130, negatively regulate cell proliferation and modulate fundamental biological processes including embryonic development, differentiation, homeostatic tissue renewal, and defense against cancer. The large, multidomain pocket proteins act by binding a plethora of cell fate-determining and growth-stimulatory proteins, the most prominent of which are the E2F/DP transcription factors. These protein-protein interactions are in turn regulated by carefully orchestrated phosphorylation events on multiple serine and threonine residues of pRB, p107, and p130, events which are carried out, at least in part, by the cyclin-dependent kinases that form the key elements of the cell cycle machinery. Here we discuss the recently obtained new insights into the diverse functions of the pRB family, and show examples of how integration of genetic, cell biology, and a range of electrophoretic approaches help to advance our understanding of the biological roles played by the pocket proteins in both normal and cancer cells.

Aberrations of p16Ink4 and retinoblastoma tumour-suppressor genes occur in distinct sub-sets of human cancer cell lines.

The p16Ink4/MTS1/CDKN2 is a cell-cycle regulatory inhibitor of cyclin-dependent kinase 4 (cdk4), and a candidate tumour suppressor whose gene on chromosome band 9p21 is frequently deleted or mutated in diverse types of cancer. Cdk4 in association with its D-type cyclin partners, together with p16Ink4, and the product of the retinoblastoma tumour-suppressor gene (pRB), appear to constitute a G1-phase-controlling pathway which can become de-regulated through oncogenic aberrations of any of the components. In an attempt to elucidate the underlying molecular mechanisms, we have now surveyed expression of p16Ink4, at the protein and the mRNA levels, in 21 human cell types expressing normal pRB, as compared with another series of 21 cell lines whose pRB is mutant and/or inactivated through sequestration by DNA tumour virus onco-proteins. In contrast to aberrant lack of p16 expression in the majority of RB-positive cell types, expression of apparently normal (as shown by electrophoretic mobility and/or the ability to form protein-protein complexes with cdk4 in vivo) p16 was uniformly preserved in the cancer cell lines whose RB function was compromised. These data indicate that p16 operates upstream of pRB along the same pathway in G1. The results are discussed in view of the nature of a selective growth advantage potentially gained by cells through de-regulation of this key cell-cycle control mechanism.

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

Soluble interleukin-2 receptor and soluble CD8 antigen levels in serum from patients with non-resectable lung cancer.

In a preliminary longitudinal study two women with histologically verified adenocarcinoma of the lung, without simultaneous infectious or inflammatory conditions, were seen every 2 weeks until death. In one of the patients serum soluble interleukin-2 receptor (sIL-2R) levels rose progressively while the levels for the other patient increased during the second half of the observation period. Serum soluble CD8 antigen (sCD8 Ag) showed a pattern dissimilar to the one for sIL-2R. In a retrospective cross-sectional study circulating levels of sIL-2R and sCD8 Ag were measured before explorative thoracotomy in a total of 65 patients with histologically proven non-resectable carcinoma of the lung. The sIL-2R levels were significantly increased independently of histological subclassification while sCD8 Ag was increased only in patients with small-cell lung cancer. There was no correlation between pre-operative values and length of survival.