Protein phohatase 1 and protein phospha-

Both cyclins A and B associate with and thereby activate cyclin‐dependent protein kinases (cdks). We have investigated which component in the cyclin‐cdk complex determines its substrate specificity. The A‐ and B‐type cyclin‐cdk complexes phosphorylated histone H1 and their cyclin subunits in an indistinguishable manner, irrespective of the catalytic subunit, p33cdk2 or p34cdc2. In contrast, only the cyclin A‐cdk complexes phosphorylated the Rb‐related p107 protein in vitro. Likewise, binding studies revealed that cyclin A‐cdk complexes bound stably to p107 in vitro, whereas cyclin B‐cdk complexes did not detectably associate with p107, under identical assay conditions. Binding to p107 required both cyclin A and a cdk as neither subunit alone bound to p107. These results demonstrate that although the kinase subunit provides a necessary component for binding, it is the cyclin subunit that plays the critical role in targeting the complex to p107. Finally, we show that the cyclin A‐p33cdk2 complex phosphorylated p107 in vitro at most of its sites that are also phosphorylated in human cells, suggesting that the cyclin A‐p33cdk2 complex is a major kinase for p107 in vivo.

Critical cell cycle transitions are controlled by the coordinate actions of the p34wc2 protein kinase and its regulatory subunits, cyclins. Recently we identified another human p34 homolog, cyclin-dependent kinase 2 (CDK2) by complementation of a cdc284 mutation in Saccharomyces cerevisiae using a AYES human cDNA expression library. CDK2 is 66% identical to CDC2Hs and 89% identical to the Xenopus Egi gene, forming a distinct subfamily of CDC2-related protein kinases. We have found that CDK2 encodes a 33-kDa cyclin A-associated protein kinase that contains phosphotyrosine, two characteristics it shares with CDC2Hs. However, we show that the subunit composition of these two protein kinase complexes can vary in different cell types, that they have different in vitro substrate preferences, and that CDK2 mRNA is observed much earlier than CDC2Hs mRNA when lymphocytes are stimulated to enter the cell cycle. We suggest that cells in different developmental or transformed states may have different mechanisms of cell cycle regulation.Critical transitions in the eukaryotic cell cycle are controlled by the coordinate action of a protein kinase(s), p34cdc2, and the associated regulatory subunits, cyclins (1-3). Originally, cyclins were defined as proteins that varied in abundance with periodicity of the cell cycle (4-7); subsequently cyclins were shown to be a family of structurally related proteins that regulate the timing of activation of p34cdc2 (8)(9)(10)(11)(12) A variety of mammalian cyclins have also been identified (5, 22-28), but except for the B-type cyclins their functions are largely unknown. The best-characterized cyclin complex is the mitotic cyclin B/p34cdc2 kinase, the active component of maturating promoting factor (5, 9-10). Cyclin A accumulates before cyclin B in the cell cycle (23) and may be involved in control of S phase (3, 23). In addition, cyclin A has been implicated in cellular transformation (27) (18) for expression in S. cerevesiae. These epitope-tagged kinases are referred to as HA-Cdk2 and HA-Cdc2Hs. Corresponding plasmids for Cdk2 and Cdc2Hs were from a previous study (17).Abbreviations: PBL, peripheral blood lymphocyte; CDK2, cyclindependent kinase 2; HA, hemagglutinin A; a-Cdk2cr, C-terminalspecific Cdk2 antibodies; a-cyclin ANT, N-terminal-specific cyclin A antibodies; a-cyclin ACHLA-1, C-terminal-specific cyclin A antibodies; RF-A, replication factor A; PHA, phytohemagglutinin-P.§To whom reprint requests should be addressed. 2907The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Basic research in genetics, biochemistry and cell biology has identified the executive enzymes and protein kinase activities that regulate the cell division cycle of all eukaryotic organisms, thereby elucidating the importance of site-specific protein phosphorylation events that govern cell cycle progression. Research in cancer genomics and virology has provided meaningful links to mammalian checkpoint control elements with the characterization of growth-promoting proto-oncogenes encoding c-Myc, Mdm2, cyclins A, D1 and G1, and opposing tumor suppressor proteins, such as p53, pRb, p16INK4A and p21WAF1, which are commonly dysregulated in cancer. While progress has been made in identifying numerous enzymes and molecular interactions associated with cell cycle checkpoint control, the marked complexity, particularly the functional redundancy, of these cell cycle control enzymes in mammalian systems, presents a major challenge in discerning an optimal locus for therapeutic intervention in the clinical management of cancer. Recent advances in genetic engineering, functional genomics and clinical oncology converged in identifying cyclin G1 (CCNG1 gene) as a pivotal component of a commanding cyclin G1/Mdm2/p53 axis and a strategic locus for re-establishing cell cycle control by means of therapeutic gene transfer. The purpose of the present study is to provide a focused review of cycle checkpoint control as a practicum for clinical oncologists with an interest in applied molecular medicine. The aim is to present a unifying model that: i) clarifies the function of cyclin G1 in establishing proliferative competence, overriding p53 checkpoints and advancing cell cycle progression; ii) is supported by studies of inhibitory microRNAs linking CCNG1 expression to the mechanisms of carcinogenesis and viral subversion; and iii) provides a mechanistic basis for understanding the broad-spectrum anticancer activity and single-agent efficacy observed with dominant-negative cyclin G1, whose cytocidal mechanism of action triggers programmed cell death. Clinically, the utility of companion diagnostics for cyclin G1 pathways is anticipated in the staging, prognosis and treatment of cancers, including the potential for rational combinatorial therapies.

GRP78 is a stress-inducible chaperone protein with antiapoptotic properties that is overexpressed in transformed cells and cells under glucose starvation, acidosis, and hypoxic conditions that persist in poorly vascularized tumors. Previously we demonstrated that the Grp78 promoter is able to eradicate tumors using murine cells in immunocompetent models by driving expression of the HSV-tk suicide gene. Here, through the use of positron emission tomography (PET) imaging, we provide direct evidence of spontaneous in vivo activation of the HSV-tk suicide gene driven by the Grp78 promoter in growing tumors and its activation by photodynamic therapy (PDT) in a controlled manner. In this report, we evaluated whether this promoter can be applied to human cancer therapy. We observed that the Grp78 promoter, in the context of a retroviral vector, was highly activated by stress and PDT in three different types of human breast carcinomas independent of estrogen receptor and p53. Complete regression of sizable human tumors was observed after prodrug ganciclovir treatment of the xenografts in immunodeficient mice. In addition, the Grp78 promoter-driven suicide gene is strongly expressed in a variety of human tumors, including human osteosarcoma. In contrast, the activity of the murine leukemia virus (MuLV) long-terminal repeat (LTR) promoter varied greatly in different human breast carcinoma cell lines, and in some cases, stress resulted in partial suppression of the LTR promoter activity. In transgenic mouse models, the Grp78 promoter-driven transgene is largely quiescent in major adult organs but highly active in cancer cells and cancer-associated macrophages, which can diffuse to tumor necrotic sites devoid of vascular supply and facilitate cell-based therapy. Thus, transcriptional control through the use of the Grp78 promoter offers multiple novel approaches for human cancer gene therapy.

Rexin-G, a nonreplicative pathology-targeted retroviral vector bearing a cytocidal cyclin G1 construct, was tested in a phase I/II study for gemcitabine-resistant pancreatic cancer. The patients received escalating doses of Rexin-G intravenously from 1 × 1011 colony-forming units (cfu) 2–3× a week (dose 0–1) to 2 × 1011 cfu 3× a week (dose 2) for 4 weeks. Treatment was continued if there was less than or equal to grade 1 toxicity. No dose-limiting toxicity (DLT) was observed, and no vector DNA integration, replication-competent retrovirus (RCR), or vector-neutralizing antibodies were noted. In nine evaluable patients, 3/3 patients had stable disease (SD) at dose 0–1. At dose 2, 1/6 patients had a partial response (PR) and 5/6 patients had SD. Median progression-free survival (PFS) was 3 months at dose 0–1, and >7.65 months at dose 2. Median overall survival (OS) was 4.3 months at dose 0–1, and 9.2 months at dose 2. One-year survival was 0% at dose 0–1 compared to 28.6% at dose 2, suggesting a dose–response relationship between OS and Rexin-G dosage. Taken together, these data indicate that (i) Rexin-G is safe and well tolerated, and (ii) Rexin-G may help control tumor growth, and may possibly prolong survival in gemcitabine-resistant pancreatic cancer, thus, earning US Food and Drug Administration's (FDA) fast-track designation as second-line treatment for pancreatic cancer.