Cell Cycle Checkpoint Inhibitors Research Articles

Abstract C204: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the Chk1/2 inhibitor, AZD7762.

Abstract Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (1–3). Here we report molecular investigations of the clinical indenoisoquinoline Top1 inhibitor, LMP-400 by itself and in combination with the cell cycle checkpoint inhibitor, AZD7762 (4). Drug effects on DNA replication and killing of cancer cells were examined. LMP-400 shows synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma HT-29 and HCT-15 cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation was similarly induced by LMP-400 and CPT and was abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) (5) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. Consistent with cell cycle checkpoint abrogation, AZD7762 inhibited both the activation of Chk1 and Chk2 autophosphorylations in LMP-400-treated cells. In spite of the potent dual inhibition of Chk1 and Chk2 at nanomolar concentrations of AZD7762, cell cycle inhibition and synergism with LMP-400 were only observed at higher concentrations and independently of Chk2 both in Chk2-complemented HT-15 and Chk2 knockout HCT-116 cells. Our study demonstrates the rationale for combining checkpoint kinase inhibitors and the novel non-camptothecin indenoisoquinoline inhibitors.

Characterization of the plasma membrane localization and orientation of HPV16 E5 for cell–cell fusion

Human papillomavirus (HPV) is a non-enveloped DNA virus with an approximately 8000 base pair genome. Infection with certain types of HPV is associated with cervical cancer, although the molecular mechanism by which HPV induces carcinogenesis is poorly understood. Three genes encoded by HPV16 are regarded as oncogenic - E5, E6, and E7. The role of E5 has been controversial. Expression of HPV16 E5 causes cell-cell fusion, an event that can lead to increased chromosomal instability, particularly in the presence of cell cycle checkpoint inhibitors like HPV16 E6 and E7. Using biochemical and cell biological assays to better understand HPV16 E5, we find that HPV16 E5 localizes to the plasma membrane with an intracellular amino terminus and an extracellular carboxyl-terminus. Further, HPV16 E5 must be expressed on both cells for cell fusion to occur. When the extracellular epitope of HPV16 E5 is targeted with an antibody, the number of bi-nucleated cells decreases.

In Vitro Mechanisms of Lovastatin on Lung Cancer Cell Lines as a Potential Chemopreventive Agent

Lung cancer causes over one million deaths per year worldwide and cigarette smoking, the proximate cause, results in a field cancerization of the respiratory track. Lung cancer cells or premalignant cells may be susceptible to apoptosis or necrosis-inducing agents. Statins inhibit the acetyl coenzyme A pathway reducing L-mevalonate that is a precursor to isoprenoids necessary for post-translational processing, resulting in apoptosis. Lovastatin was added to four lung cancer cell lines and normal human bronchial epithelial cells followed by Western blots to evaluate proteins in the cell cycle, oxidant, and apoptotic pathways. Flow cytometry revealed significant increases in three of four lung cancer cell lines in apoptosis and necrosis after lovastatin treatment at 10 microM for 72 h. Lovastatin adversely affected lung cancer cell survival with increases in cell-cycle check-point inhibitors p21WAF and/or p27KIP and a decrease in cyclin D1. All four lung cancer cell lines had a decrease in glutathione after lovastatin treatment consistent with reduced protection against reactive oxidant species. Three of four lung cancer cell lines had increased cytochrome c release with reduced pro-caspase-3 and increases in activated caspase-3. Lovastatin induces apoptosis and necrosis in lung cancer cell lines by causing alterations in the cell cycle, reducing glutathione, and activating p53, Bax protein, and caspases while increasing cytochrome c in apoptosis pathways. Targeting HMG-CoA reductase may represent an approach to lung cancer chemotherapy, e.g., reversing ground glass opacities detected on CT scans or resolving airway preneoplasias detected by bronchoscopy before they progress to malignant transformation.

Human lipodystrophies linked to mutations in A-type lamins and to HIV protease inhibitor therapy are both associated with prelamin A accumulation, oxidative stress and premature cellular senescence

Lipodystrophic syndromes associated with mutations in LMNA, encoding A-type lamins, and with HIV antiretroviral treatments share several clinical characteristics. Nuclear alterations and prelamin A accumulation have been reported in fibroblasts from patients with LMNA mutations and adipocytes exposed to protease inhibitors (PI). As genetically altered lamin A maturation also results in premature ageing syndromes with lipodystrophy, we studied prelamin A expression and senescence markers in cultured human fibroblasts bearing six different LMNA mutations or treated with PIs. As compared to control cells, fibroblasts with LMNA mutations or treated with PIs had nuclear shape abnormalities and reduced proliferative activity that worsened with increasing cellular passages. They exhibited prelamin A accumulation, increased oxidative stress, decreased expression of mitochondrial respiratory chain proteins and premature cellular senescence. Inhibition of prelamin A farnesylation prevented cellular senescence and oxidative stress. Adipose tissue samples from patients with LMNA mutations or treated with PIs also showed retention of prelamin A, overexpression of the cell cycle checkpoint inhibitor p16 and altered mitochondrial markers. Thus, both LMNA mutations and PI treatment result in accumulation of farnesylated prelamin A and oxidative stress that trigger premature cellular senescence. These alterations could participate in the pathophysiology of lipodystrophic syndromes and lead to premature ageing complications.

Pharmacological Abrogation of S-Phase Checkpoint Enhances the Anti-Tumor Activity of Gemcitabine In Vivo

Chk1 and Chk2 kinases are critically involved in modulating DNA damage checkpoints. In particular, Chk1, a key activator of the S-phase DNA damage response, may be involved in resistance to genotoxic therapies that target DNA synthesis. We studied the in vitro and in vivo effects of EXEL-9844 (XL844), a potent, orally available, and specific inhibitor of Chk1 and Chk2, in combination with gemcitabine. In clonogenic assays using multiple cell lines in vitro, EXEL-9844 had only minor effects as a single agent but substantially enhanced gemcitabine-induced cell killing. Correspondingly, in PANC-1 cells, EXEL-9844 increased gemcitabine-induced H2AX phosphorylation, blocked Cdc25A phosphorylation, and induced premature mitotic entry. In a PANC-1 xenograft model, EXEL-9844 significantly enhanced gemcitabine antitumor activity but had limited effect as a single agent. Together, these data show that cell cycle checkpoint inhibitors may have significant clinical utility in potentiating the activity of gemcitabine.

P53-based cancer therapies: is defective p53 the Achilles heel of the tumor?

The tumor suppressor protein p53 plays a pivotal role in the DNA damage response and is defective in >50% of human tumors, which has generated substantial interest in developing p53-targeted cancer therapies. Various therapeutic rationales targeting p53 are currently under investigation including attempts to both activate and inhibit p53. Elevation of p53 can be achieved by either reintroducing an exogenous p53 gene or by blocking its association with its negative regulator hDM2. An alternate approach involves reverting mutant p53 to its wild-type conformation. Inhibition of p53 activity can be achieved either by preventing p53-mediated gene expression or by inhibiting the mitochondrial pro-apoptotic interactions of p53. These approaches are based on the concept that activation of p53 in a tumor is cytotoxic while inhibition of p53 in normal cells will protect the patient. However, activation of p53 also induces cell cycle arrest that can protect most normal cells from DNA damage, and this is the reason why many p53-defective tumors are more sensitive to DNA damage. The development of cell cycle checkpoint inhibitors to abrogate DNA damage-induced arrest builds on this observation as p53-defective cells appear particularly sensitive. Thus, normal cells are protected from premature entry into mitosis and the subsequent mitotic catastrophe induced by checkpoint inhibitors, while p53-defective tumor cells are destroyed. These contradictory approaches must be resolved if we are to take full advantage of the frequent p53 defect in tumors.

A pathway regulated by cell cycle inhibitor p27Kip1 and checkpoint inhibitor Smad3 is involved in the induction of T cell tolerance

Peripheral tolerance is essential for immunological homeostasis. Tolerant T cells are thought to arise after T cell receptor ligation in conditions that are nonpermissive for replication. Here we have investigated the function of the cell cycle inhibitor p27(Kip1) in tolerance induction in vivo using naive T cell receptor-transgenic cells lacking the cyclin-dependent kinase (Cdk)-binding domain of p27(Kip1)(p27delta). Wild-type but not p27delta cells underwent tolerization. Tolerized wild-type cells had impaired Cdk2 and Cdc2 kinase activity and failed to phosphorylate the checkpoint inhibitor Smad3, leading to enhanced expression of the Cdk inhibitor p15. In contrast, p27delta cells proliferated in tolerizing conditions because of Cdk kinase activation and phosphorylation of Smad3, which resulted in no upregulation of p15. Smad3 'knockdown' prevented tolerance induction, whereas expression of a Smad3 mutant resistant to Cdk-mediated phosphorylation recapitulated molecular and functional events of tolerance. Thus, p27(Kip1) is required during induction of tolerance and Smad3 regulates T cell responses 'downstream' of p27(Kip1).

Total Synthesis of Ustiloxin D and Considerations on the Origin of Selectivity of the Asymmetric Allylic Alkylation.

As part of investigations into cell cycle checkpoint inhibitors, an asymmetric synthesis of the antimitotic natural product, ustiloxin D, has been completed. A salen-Al-catalyzed aldol reaction was employed to construct a chiral oxazoline 9 (99% yield, 98% ee) that served the dual purpose of installing the necessary 1,2-amino alcohol functionality as well as providing an efficient synthon for the requisite methylamino group at C9. The chiral aryl-alkyl ether was assembled using a Pd-catalyzed asymmetric allylic alkylation that notably delivered a product with stereochemistry opposite to that predicted by precedent. The linear tetrapeptide was subsequently cyclized to produce ustiloxin D. The mechanistic origin of the allylic alkylation selectivity was further investigated, and a working hypothesis for the origin of the observed stereoselectivity has been proposed.

Total Synthesis of Ustiloxin D and Considerations on the Origin of Selectivity of the Asymmetric Allylic Alkylation

As part of investigations into cell cycle checkpoint inhibitors, an asymmetric synthesis of the antimitotic natural product, ustiloxin D, has been completed. A salen-Al-catalyzed aldol reaction was employed to construct a chiral oxazoline 9 (99% yield, 98% ee) that served the dual purpose of installing the necessary 1,2-amino alcohol functionality as well as providing an efficient synthon for the requisite methylamino group at C9. The chiral aryl-alkyl ether was assembled using a Pd-catalyzed asymmetric allylic alkylation that notably delivered a product with stereochemistry opposite to that predicted by precedent. The linear tetrapeptide was subsequently cyclized to produce ustiloxin D. The mechanistic origin of the allylic alkylation selectivity was further investigated, and a working hypothesis for the origin of the observed stereoselectivity has been proposed.

Granulatimide and Isogranulatimide, Aromatic Alkaloids with G2 Checkpoint Inhibition Activity Isolated from the Brazilian Ascidian Didemnum granulatum: Structure Elucidation and Synthesis

Crude methanol extracts of the ascidian Didemnum granulatum collected in Brazil showed activity in a new screen for G2 cell cycle checkpoint inhibitors. Bioassay-guided fractionation of the extract yielded the known alkaloids didemnimides A (1) and D (2), the new alkaloid didemnimide E (3), and a new G2 checkpoint inhibitor. Two candidate structures for the inhibitor, named granulatimide (4) and isogranulatimide (5), have been prepared via a short and efficient biomimetic synthesis involving the photolysis of didemnimide A (1). The synthesis revealed that the correct structure for the naturally occurring G2 checkpoint inhibitor is isogranulatimide (5). Granulatimide (4), the other candidate structure, was also found to be a G2 checkpoint inhibitor, and it was subsequently detected in chromatographic fractions associated with purification of D. granulatum alkaloids. Granulatimide (4) and isogranulatimide (5) represent the first examples of a new class of G2 specific cell cycle checkpoint inhibitors and the...