Checkpoint genes in cancer.

The mammalian cell cycle is exquisitely controlled by a 'machinery' composed of cyclin-dependent kinases and their binding partners, the cyclins. These kinases regulate transitions into DNA synthesis and mitosis, and their inactivity contributes to cellular quiescence, differentiation and senescence. Cell cycle transitions are, in turn, controlled by checkpoints that monitor ribonucleotide pools, oxygen tension, the extracellular environment, growth signalling programmes, the status of DNA replication, and the mitotic spindle apparatus. Genes positively controlling cell cycle checkpoints can be targets for oncogenic activation in cancer, whereas negative regulators, such as tumour suppressor genes, are targeted for inactivation. Understanding the molecular details of cell cycle regulation and checkpoint abnormalities in cancer offers insight into potential therapeutic strategies.

Death domain mutagenesis of KILLER/DR5 reveals residues critical for apoptotic signaling.

The Fas/tumor necrosis factor (TNF)/TRAIL receptors signal death through a cytoplasmic death domain (DD) containing six alpha-helices with positively charged helix 2 interacting with negatively charged helix 3 of another DD. DD mutation occurs in head/neck and lung cancer (TRAIL receptor KILLER/DR5) and in lpr mice (Fas). We examined the apoptotic potential of known KILLER/DR5 lung tumor-derived mutants (n = 6) and DD mutants (n = 18) generated based on conservation with DR4, Fas, Fas-associated death domain (FADD), and tumor necrosis factor receptor 1 (TNFR1). With the exception of Arg-330 required in Fas or FADD for aggregation or for TNFR1 cytotoxicity, surprisingly major loss-of-function KILLER/DR5 alleles (W325A, L334A (lpr-like), I339A, and W360A) contained hydrophobic residues. Loss-of-function of I339A (highly conserved) has not been reported in DDs. Charged residue mutagenesis revealed the following points. 1) E326A, conserved in DR4, is dispensable for death; the homologous residue is positively charged in Fas, TNFR1, and FADD and is critical for DD interactions. 2) K331A, D336A, E338A, K340A, K343A, and D351A have partial loss-of-function suggesting multiple charges stabilize receptor-adapter interactions. Analysis of the tumor-derived KILLER/DR5 mutants revealed the following. 1) L334F has partial loss-of-function versus L334A, whereas E338K has major loss-of-function versus E338A, examples where alanine and tumor-specific substitutions have divergent phenotypes. 2) Unexpectedly, S324F, E326K, K386N, and D407Y have no loss-of-function with tumor-specific or alanine substitutions. Loss-of-function KILLER/DR5 mutants were deficient in recruitment of FADD and caspase 8 to TRAIL death-inducing signaling complexes. The results reveal determinants within KILLER/DR5 for death signaling and drug design.

The TRAIL decoy receptor TRUNDD (DcR2, TRAIL-R4) is induced by adenovirus-p53 overexpression and can delay TRAIL-, p53-, and KILLER/DR5-dependent colon cancer apoptosis.

The cell surface decoy receptor proteins TRID (also known as DcR1 or TRAIL-R3) and TRUNDD (DcR2, TRAIL-R4) inhibit caspase-dependent cell death induced by the cytotoxic ligand TRAIL in part because of their absent or truncated cytoplasmic death domains, respectively. We previously identified the death domain containing proapoptotic TRAIL death receptor KILLER/DR5 (TRAIL-R2) as an upregulated transcript following exposure of cancer cells, with wild-type but not with mutant or degraded p53 proteins, to a cytotoxic dose of adriamycin. In the present studies we provide evidence that expression of the TRAIL decoy receptors TRUNDD and TRID increases following infection of cancer cells with p53-expressing adenovirus (Ad-p53), in a manner similar to other p53 target genes such as KILLER/DR5 and p21WAF1/CIP1. Subsequent overexpression of TRUNDD in colon cancer cell lines caused a significant delay in killing induced by TRAIL. Furthermore, cotransfection of TRUNDD with either p53 or KILLER/DR5 (at a 4:1 DNA ratio) in colon cancer cells decreased cell death caused by either gene. This protective effect of TRUNDD was not dependent on the presence of TRAIL, and overexpression of TRUNDD did not alter the protein levels of either p53 or KILLER/ DR5. Further deletion studies showed that whereas protection by TRUNDD against TRAIL-mediated apoptosis did not require an intact intracellular domain (ICD), the first 43 amino acids of the ICD of TRUNDD were needed for protection against cell death induced by p53 or KILLER/DR5. Our results suggest a model in which the TRAIL decoy receptors may be induced by p53, thereby attenuating an apoptotic response that appears to involve KILLER/DR5. Therefore, the p53-dependent induction of TRUNDD may provide a mechanism to transiently favor cell survival over cell death, and overexpression of TRUNDD may be another mechanism of escape from p53-mediated apoptosis in gene therapy experiments.

Cell cycle control as a basis for cancer drug development (Review).

Normal cell cycle progression relies on the cell's ability to translate extracellular signals such as mitogenic stimuli and intact extracellular matrices in order to efficiently replicate DNA and divide. Cyclin dependent kinases (cdks) respond to these signals and are largely responsible for positively pushing cells through the cell cycle. Due to their pivotal role in cell division, nature has evolved elaborate mechanisms to regulate the kinase activity of cdks. Cyclins are cdk binding partners which are required for kinase activity and their protein levels are intimately linked to the cell cycle stage. A variety of other cdk regulators such as phosphorylation events, natural inhibitors and complex stability are discussed. Phosphorylation of various cdk substrates results in diverse outcomes such as changes in gene expression, formation of prereplicative complexes and breakdown of the nuclear envelope. Cancer cells evolve in part by over-riding normal cell cycle regulation. Abnormal cdk activity is accomplished by cyclin amplification, cdk or substrate mutation as well as inactivation of inhibitors. The selective growth advantage of cancer cells also stems from amplification of positive growth signals, mutation of checkpoint and surveillance genes as well as deregulation of programmed cell death or apoptosis. The full potential of cancer therapies, such as small molecule inhibitors and gene therapy among others, focusing on our knowledge of cell cycle regulation has yet to be reached.

Induction of the TRAIL receptor KILLER/DR5 in p53-dependent apoptosis but not growth arrest.

The TRAIL death receptor KILLER/DR5 is induced by DNA damaging agents in wild-type p53-expressing cells. Here we show that, unlike the p53-target CDK-inhibitor p21WAF1/CIP1, the TRAIL death receptor KILLER/DR5 is only induced in cells undergoing p53-dependent apoptosis and not cell cycle arrest. Thus GM glioblastoma cells carrying an inducible MMTV-driven p53 gene undergo cell cycle arrest and upregulate p21 but not KILLER/DR5 expression upon dexamethasone exposure. WI38 normal lung fibroblasts undergoing cell cycle arrest in response to ionizing irradiation also induce p21 but not KILLER/DR5 gene expression. KILLER/DR5 upregulation is also deficient in irradiated lymphoblastoid cells derived from patients with Ataxia Teleangiectasia suggesting a role for the ATM-p53 pathway in regulating KILLER/DR5 expression after DNA damage. Inhibition of transcription by Actinomycin D blocks both KILLER/DR5 and p21 induction in cells undergoing p53-dependent apoptosis. Our results suggest that the p53-dependent transcriptional induction of KILLER/DR5 death receptor is restricted to cells undergoing apoptosis and not cells undergoing exclusively p53-dependent G1 arrest.

Repair Defect in p21 WAF1/CIP1 -/- human cancer cells.

p53 induction and cell cycle arrest occur following DNA damage, possibly to allow repair prior to replication. p21WAF1/CIP1, a cyclin-cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen-interacting protein, is induced by p53 and mediates the cell cycle arrest. To investigate a role for p21 in DNA repair in vivo, we studied the expression of in vitro damaged reporter DNA transfected into p21 +/+ or -/- HCT116 human colon cancer cells. Introduction of UV-damaged or cisplatinum-damaged cytomegalovirus-driven beta-galactosidase reporter DNA into tumor cells revealed a significant decrease (2-5-fold) in reporter expression in p21 -/- versus +/+ cells. In the absence of DNA damage, there was a significant increase (2-3-fold) in the number of 6-TG-resistant colonies derived from p21 -/- versus +/+ cells. Reintroduction of wild-type p21, but not a p21 C-terminal truncation mutant which lacks the proliferating cell nuclear antigen interaction domain, stimulated (2-3-fold) the repair capacity of the p21-deficient cells. We conclude that p21 deficiency is associated with a defect in DNA repair, which could lead to an increased sensitivity of tumor cells to DNA damage.

Evaluation of the psychological status of ventilatory-supported patients with ALS/MND.

Many medical professionals feel that a choice of long-term ventilatory support leads to a life of hopeless desperation. We compared the sociodemographic, physical and psychological status of 18 amyotrophic lateral sclerosis/motor neurone disease (ALS/MND) patients on ventilatory support for 1 to 120 months with that of 126 nonventilatory-supported ALS/MND patients. Patients filled out a comprehensive data form and completed ten psychological tests. A composite psychological status score was computed, representing a continuum from psychological distress to psychological well-being. Mann-Whitney and chi 2 tests were used to compare the two groups. There were no significant differences in sociodemographic makeup, depression, hopelessness, overall quality of life or psychological well-being. However, ventilatory-supported patients had a more internal health locus of control. Many patients on ventilatory support were able to live high quality lives. When ventilatory support is an option, we suggest that medical professionals be supportive of the patient's choices and recognise that a decision for ventilatory support is probably the best predictor of an acceptable quality of life on a ventilator.

Survival in amyotrophic lateral sclerosis. The role of psychological factors.

OBJECTIVE: Examining the relationship between psychological status and survival in amyotrophic lateral sclerosis. Our hypothesis is that psychological distress is associated with greater mortality and shorter survival time than psychological well-being. DESIGN: Cross-sectional, longitudinal. The baseline evaluations used were disease severity and 10 psychometric tests. A psychological status score was derived from these tests. Survival status was monitored for 3.5 years. Interviewers were blinded to other interviews and data analysis. SETTING: Patient's residence. PATIENTS: The criteria for eligibility were diagnosis of amyotrophic lateral sclerosis by a neurologist, dementia or alcoholism absent, communication in English, and any severity or length of disease. It was a volunteer sample consisting of 144 patients from amyotrophic lateral sclerosis clinics or community-based amyotrophic lateral sclerosis support groups. In this sample 66% were men, 94% were white, mean age at diagnosis was 55 years, 79% were married, 60% had some college education, and 61% died during the study. INTERVENTIONS: None. END POINTS: mortality during study, survival time from intake to last follow-up. RESULTS: Comparison between high and low psychological score groups: 32% of high and 82% of low died; survival curves were significantly different. Controlling for confounding factors (length of illness, disease severity, age), patients with psychological distress had a greater risk of mortality (relative risk, 6.76; 95% confidence limits, 1.69 to 27.12) and greater likelihood of dying in any given time period (relative risk, 2.24; 95% confidence limits, 1.08 to 4.64) than those with psychological well-being. CONCLUSION: Adjusting for confounding factors, psychological status is strongly related to outcome in amyotrophic lateral sclerosis. Further studies on psychological status should be done to confirm its prognostic value.