Management and survival trends in advanced colorectal cancer.

AIMS: Significant improvements in the outcome for patients with advanced colorectal cancer (CRC) have been achieved. The median survival for advanced CRC reported in clinical trials now approaches 2 years, but there is often a question as to whether this partly represents patient selection. We aimed to explore whether the availability of new chemotherapy drugs (irinotecan and oxaliplatin) and surgical advances have affected survival in a normal clinical setting. MATERIALS AND METHODS: A review of the Queen Elizabeth and Lyell McEwin health service prospective CRC database from 1992 to 2004 was carried out to assess outcome differences between two time cohorts (1 January 1992-31 December 1997 and 1 January 1998-31 December 2004). RESULTS: For all patients (n = 744) overall survival was seen to improve over time and is maintained out to 5 years. There have been a number of trends over time (1992-1997 vs 1998-2004) that have probably contributed to this gain; increased overall chemotherapy use (33% vs 43%); use of combination chemotherapy (i.e. oxaliplatin and irinotecan regimens); increased hepatic resection rates (1.9% vs 10.8%) and increased clinical trial uptake (0.6% vs 14.5%). CONCLUSION: This current analysis confirms an improvement in survival over time for advanced CRC and this is seen in unselected patients including those over 70 years of age.

Sequence-specific binding of Ku autoantigen to single-stranded DNA.

Glucocorticoid-induced transcription of mouse mammary tumor virus is repressed by Ku antigen/DNA-dependent protein kinase (DNA-PK) through a DNA sequence element (NRE1) in the viral long terminal repeat. Nuclear factors binding to the separated single strands of NRE1 have been identified that may also be important for transcriptional regulation through this element. We report the separation of the upper-stranded NRE1 binding activity in Jurkat T cell nuclear extracts into two components. One component was identified as Ku antigen. The DNA sequence preference for Ku binding to single-stranded DNA closely paralleled the sequence requirements of Ku for double-stranded DNA. Recombinant Ku bound the single, upper strand of NRE1 with an affinity that was 3-4-fold lower than its affinity for double-stranded NRE1. Sequence-specific single-stranded Ku binding occurred rapidly (t1/2 on = 2.0 min) and was exceptionally stable, with an off rate of t1/2= 68 min. While Ku70 cross-linked to the upper strand of NRE1 when Ku was bound to double-stranded and single-stranded DNAs, the Ku80 subunit only cross-linked to single-stranded NRE1. Intriguingly, addition of Mg2+ and ATP, the cofactors required for Ku helicase activity, induced the cross-linking of Ku80 to a double-stranded NRE1-containing oligonucleotide, without completely unwinding the two strands.

Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor.

NRE1 is a DNA sequence element through which Ku antigen/DNA-dependent protein kinase (DNA-PK) catalytic subunit represses the induction of mouse mammary tumor virus transcription by glucocorticoids. Although Ku is an avid binder of DNA ends and has the ability to translocate along DNA, we report that direct sequence-specific Ku binding occurs with higher affinity (Kd = 0.84 +/- 0.24 nM) than DNA end binding. Comparison of Ku binding to several sequences over which Ku can accumulate revealed two classes of sequence. Sequences with similarity to NRE1 competed efficiently for NRE1 binding. Conversely, sequences lacking similarity to NRE1 competed poorly for Ku and were not recognized in the absence of DNA ends. Phosphorylation of glucocorticoid receptor (GR) fusion proteins by DNA-PK reflected Ku DNA-binding preferences and demonstrated that co-localization of GR with DNA-PK on DNA in cis was critical for efficient phosphorylation. Phosphorylation of the GR fusion protein by DNA-PK mapped to a single site, Ser-527. This site occurs adjacent the GR nuclear localization sequence between the DNA and ligand binding domains of GR, and thus its phosphorylation, if confirmed, has the potential to affect receptor function in vivo.

Sequence-specific DNA binding by Ku autoantigen and its effects on transcription.

DNA-dependent protein kinase (DNA-PK) has been implicated in several nuclear processes including transcription, DNA replication, double-stranded DNA break repair, and V(D)J recombination. Linkage of kinase and substrate on DNA in cis is required for efficient phosphorylation. Recruitment of DNA-PK to DNA is by Ku autoantigen, a DNA-end-binding protein required for DNA-PK catalytic activity. Although Ku is known to translocate along naked DNA, how DNA-end binding by Ku might lead to DNA-PK-mediated phosphorylation of sequence-specific DNA-binding proteins in vivo has not been obvious. Here we report the identification of Ku as a transcription factor that recruits DNA-PK directly to specific DNA sequences. NRE1 (negative regulatory element 1) is a DNA sequence element (-394/ -381) in the long terminal repeat of mouse mammary tumour virus (MMTV) that is important for repressing inappropriate viral expression. We show that direct binding of Ku/DNA-PK to NRE1 represses glucocorticoid-induced MMTV transcription.

Multi-strand binding of nuclear factors to a repressor of mouse mammary tumor virus transcription can be distinguished kinetically.

NRE1 is a DNA sequence element in the long terminal repeat of mouse mammary tumor virus that represses viral transcription in mature T cells. In addition to double-stranded binding activity, factors in Jurkat T cell nuclear extracts bind specifically to each of the two single-strands of NRE1. Here we show that binding to the three forms of NRE1 can be distinguished kinetically. The on rates for double, upper and lower-strand NRE1 binding were 1.5, 3, and 11 min, respectively. Binding was extremely stable with off-rates varying from 30 and 60 min for double and upper-strand binding to 12 h for lower-strand binding. In addition, a truncated form of NRE1 that is only bound as a double-strand was observed to have an on rate of binding of 4 min and an off rate of 4 h.

Poly(vinyl alcohol) as a blocking agent in enzyme immunoassays.

We examined the blocking ability of poly(vinyl alcohol) (PVA) in enzyme immunoassays by coating polystyrene microtiter wells with PVA of different molecular weights (MW) and percent hydrolysis (%Hyd). Blocking ability was measured by the differences in non-specific binding of an anti-rabbit IgG-horseradish peroxidase conjugate to coated and uncoated wells. PVA with a MW of 124,000-186,000 and > 99 %Hyd was the most effective in suppressing the binding of the conjugate. This PVA at 0.5% (w/v) was significantly better at reducing non-specific binding than commonly used blocking agents and did not interfere with the specific binding of the conjugate to antigen-coated microtiter wells.

Measuring the precision of genetic parameters by a simulation technique.

Approximate standard errors of genetic parameter estimates were obtained using a simulation technique and approximation formulae for a simple statistical model. The similarity of the corresponding estimates of standard errors from the two methods indicated that the simulation technique may be useful for estimating the precision of genetic parameter estimates for complex models or unbalanced population structures where approxi mation formulae do not apply. The method of generating simulation populations in the computer is outlined, and a technique of setting approximate confidence limits to heritability estimates is described.