Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Cell Cycle Proteins as Promising Targets in Cancer Therapy

Cyclin-dependent kinases (cdks) are critical regulators of cell cycle progression and RNA transcription. A variety of genetic and epigenetic events cause universal overactivity of the cell cycle cdks in human cancer, and their inhibition can lead to both cell cycle arrest and apoptosis. However, built-in redundancy may limit the effects of highly selective cdk inhibition. Cdk4/6 inhibition has been shown to induce potent G1 arrest in vitro and tumor regression in vivo; cdk2/1 inhibition has the most potent effects during the S and G2 phases and induces E2F transcription factor-dependent cell death. Modulation of cdk2 and cdk1 activities also affects survival checkpoint responses after exposure to DNA-damaging and microtubule-stabilizing agents. The transcriptional cdks phosphorylate the carboxy-terminal domain of RNA polymerase II, facilitating efficient transcriptional initiation and elongation. Inhibition of these cdks primarily affects the accumulation of transcripts with short half-lives, including those encoding antiapoptosis family members, cell cycle regulators, as well as p53 and nuclear factor-kappa B-responsive gene targets. These effects may account for apoptosis induced by cdk9 inhibitors, especially in malignant hematopoietic cells, and may also potentiate cytotoxicity mediated by disruption of a variety of pathways in many transformed cell types. Current work is focusing on overcoming pharmacokinetic barriers that hindered development of flavopiridol, a pan-cdk inhibitor, as well as assessing novel classes of compounds potently targeting groups of cell cycle cdks (cdk4/6 or cdk2/1) with variable effects on the transcriptional cdks 7 and 9. These efforts will establish whether the strategy of cdk inhibition is able to produce therapeutic benefit in the majority of human tumors.

Cyclin-dependent kinase pathways as targets for cancer treatment.

In the last decade, the discovery and cloning of the cyclin-dependent kinases (cdks), key regulators of cell cycle progression, have led to the identification of novel modulators of cdk activity. Initial experimental results demonstrated that these cdk modulators are able to block cell cycle progression, induce apoptotic cell death, promote differentiation, inhibit angiogenesis, and modulate transcription. Alteration of cdk activity may occur indirectly by affecting upstream pathways that regulate cdk activity or directly by targeting the cdk holoenzyme. Two direct cdk modulators, flavopiridol and UCN-01, are showing promising results in early clinical trials, in which the drugs reach plasma concentrations that can alter cdk activity in vitro. Although modulation of cdk activity is a well-grounded concept and new cdk modulators are being assessed for clinical testing, important scientific questions remain to be addressed. These questions include whether one or more cdks should be inhibited, how cdk inhibitors should be combined with other chemotherapy agents, and which cdk substrates should be used to assess the biologic effects of these drugs in patients. Thus, modulation of cdk activity is an attractive target for cancer chemotherapy, and several agents that modulate cdk activity are in or are approaching entry into clinical trials.

Preclinical and Clinical Development of Cyclin-Dependent Kinase Modulators

A significant number of drugs and drug candidates in clinical development are halogenated structures. For a long time, insertion of halogen atoms on hit or lead compounds was predominantly performed to exploit their steric effects, through the ability of these bulk atoms to occupy the binding site of molecular targets. However, halogens in drug - target complexes influence several processes rather than steric aspects alone. For example, the formation of halogen bonds in ligand-target complexes is now recognized as a kind of intermolecular interaction that favorably contributes to the stability of ligand-target complexes. This paper is aimed at introducing the fascinating versatility of halogen atoms. It starts summarizing the prevalence of halogenated drugs and their structural and pharmacological features. Next, we discuss the identification and prediction of halogen bonds in protein-ligand complexes, and how these bonds should be exploited. Interesting results of halogen insertions during the processes of hit-to-lead or lead-to-drug conversions are also detailed. Polyhalogenated anesthetics and protein kinase inhibitors that bear halogens are analyzed as cases studies. Thereby, this review serves as one guide for the virtual screening of libraries containing halogenated compounds and may be a source of inspiration for the medicinal chemists.

Halogen atoms in the modern medicinal chemistry: hints for the drug design.

Apoptosis and proliferation are intimately coupled. Some cell cycle regulators can influence both cell division and programmed cell death. The linkage of cell cycle and apoptosis has been recognized for c-Myc, p53, pRb, Ras, PKA, PKC, Bcl-2, NF-kappa B, CDK, cyclins and CKI. This review summarizes the different functions of the proteins presently known to control both apoptosis and cell cycle progression. These proteins can influence apoptosis or proliferation but different variables, including cell type, cellular environment and genetic background, make it difficult to predict the outcome of cell proliferation, cell cycle arrest or cell death. These important decisions of cell proliferation or cell death are likely to be controlled by more than one signal and are necessary to ensure a proper cellular response.

/pdf/cell-cycle-and-apoptosis-2rd0h42jzr.pdf

Cell cycle and apoptosis.

Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes Immunosuppression, and Has Potent Antitumor Activity In Vivo Against Human Leukemia and Lymphoma Xenografts

Cytotoxicity and apoptosis of benzoquinones: redox cycling, cytochrome c release, and BAD protein expression.

The elevated expression of the flavoprotein NAD(P)H:quinone acceptor oxidoreductase (NQO1) (EC 1.6.99.2) in many human solid tumors, along with its ability to activate quinone-based anticancer agents, makes it an excellent target for enzyme-directed drug development. Previous studies have shown a significant statistical correlation between NQO1 enzymatic activity and the cytotoxicity of certain antitumor quinones. RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], presently in late preclinical and entering early clinical development, has been previously considered to be an excellent substrate for activation by NQO1. In this study we investigate the cytotoxicity of RH1 in cell lines selected from the NCI's 60 tumor cell line panel, expressing varying levels of NQO1 activity. Exposure time- and concentration-dependent cytotoxicity was seen, apparently independent from levels of NQO1 activity in these cells. Furthermore, the NQO1 inhibitor dicoumarol had no impact on the sensitivity profiles of RH1 response. The HL-60 myeloid leukemia cells, which do not have detectable NQO1 activity, were further investigated. RH1 treatment of HL-60 cells generated high levels of free radicals, which was accompanied by robust redox cycling, oxygen consumption and induction of apoptosis. These results are in agreement with previous data suggesting that, in addition to its activation by NQO1, RH1-induced cytotoxicity might involve alternative pathways for activation of this compound. Furthermore, the high cytotoxicity of RH1 in the leukemia/lymphoma subpanel of the NCI in vitro cell line screen would suggest an empirical rationale for the utilization of this compound in the treatment of these malignancies.

Cytotoxicity of RH1: NAD(P)H:quinone acceptor oxidoreductase (NQO1)-independent oxidative stress and apoptosis induction.

PCR-based fluorescent detection assays for the relative and quantitative measurement of gene expression, such as Taq Man™, LUX™, and SYBR Green™, are currently in wide spread use due to their general applicability, low cost, reproducibility, accuracy, and ease of use. One current limitation of quantitative PCR (Q-PCR) is the lack of a fully integrated and high-throughput method for general genomic and diagnostic applications. Here we report a reliable and high-throughput system for the automated extraction of RNA, first-strand cDNA synthesis, quality control measures, consecutive real-time PCR amplification, and primary data analysis. As described, this procedure utilizes commonly available reagents and pre-packaged “kits” for RNA extraction, first strand cDNA synthesis, Q-PCR, liquid handling, and capillary electrophoresis that are generally applicable to a wide variety of robotic platforms. (JALA 2004;9:128-34)

https://journals.sagepub.com/doi/pdf/10.1016/j.jala.2004.04.011

Gabriela Tudor

Papers

Flavopiridol Induces Apoptosis of Normal Lymphoid Cells, Causes Immunosuppression, and Has Potent Antitumor Activity In Vivo Against Human Leukemia and Lymphoma Xenografts

Cytotoxicity and apoptosis of benzoquinones: redox cycling, cytochrome c release, and BAD protein expression.

Cytotoxicity of RH1: NAD(P)H:quinone acceptor oxidoreductase (NQO1)-independent oxidative stress and apoptosis induction.

Automated Process of Q-PCR/Gene Expression