Review article: strategies for the management of chronic unremitting ulcerative colitis.

BACKGROUND: Chronic active ulcerative colitis (UC) is associated with significant morbidity, loss of productivity, increased colorectal cancer risk and cost. Up to 18% of patients suffer chronic active disease, with 30% requiring colectomy at 10 years. The management remains challenging given the relatively few clinical trials in this area. AIM: To summarise the evidence regarding optimal management strategies for patients with chronic active UC of differing disease extents and degrees of treatment refractoriness. METHOD: A literature search using the PubMed and Medline databases was performed. No time limit was set on article publication for inclusion. RESULTS: The principles of management should focus on confirming disease activity, exclusion of alternative diagnoses, adherence and treatment escalation. Infliximab and topical tacrolimus are options in refractory proctitis, although the evidence for these therapies is limited. Both infliximab and adalimumab are effective in corticosteroid-refractory disease, although the proportions of patients achieving corticosteroid-free remission remain modest (24% at 30 weeks and 16.9% at 8 weeks respectively). Alternatives include ciclosporin and tacrolimus, and possibly methotrexate. Colectomy often leads to an improved quality of life; medical strategies unlikely to provide durable corticosteroid-free remission should not be pursued. CONCLUSIONS: No current pharmacological treatment delivers mucosal healing in the majority of patients. Newer treatments such as vedolizumab and tofacitinib may represent valuable future therapies. Available medical options should be discussed with patients at every step of their management, with an honest appraisal of the evidence. Surgery should always be considered in patients with chronic refractory disease of any extent.

On target? Strategies and progress in the development of therapies for colorectal cancer targeted against WNT signalling.

Chemotherapy is an integral part of treatment for colorectal cancer (CRC), especially in the context of metastatic cancer. Targeted therapies in the form of monoclonal antibodies directed against the endothelial growth factor receptor or the vascular endothelial growth factor have met with success, and have demonstrated the advantages of molecularly targeted therapy in colorectal cancer. Nevertheless, CRC remains a major cause of death, which demonstrates the urgent need for improved treatment strategies. The pathway activated by the Wingless-type mouse mammary tumour virus integration site (WNT) family members is constitutively active and promotes cancer growth in the majority of CRCs. As a result, there has been interest in developing therapeutics that circumvent it either by inhibiting WNT-mediated transcription or by inactivating the target genes. This review considers the current therapies approved for use in CRC and discusses the progress with therapies designed to target the WNT signalling pathway.

Human papillomavirus infection and anal dysplasia in renal transplant recipients.

BACKGROUND: Immunosuppression is a known risk factor for anal human papillomavirus (HPV) disease, including anal squamous cell carcinoma. Additional risk factors for HPV-related disease have not been studied in the renal transplant population. The demographics of anal HPV and associated risk factors were investigated in this population. METHODS: Anal cytology and polymerase chain reaction were used to assess anal HPV disease in a cohort of transplant recipients at the Royal London Hospital. Risk factors associated with increased immunosuppression and HPV exposure were collated to determine any association with anal disease. RESULTS: Anal dysplasia was associated with anal oncogenic HPV infection (P < 0.001), duration of immunosuppression (P = 0.050), previous genital warts (P = 0.018) and receptive anal intercourse (P = 0.013). CONCLUSION: Anal dysplasia was related to immunosuppression and patient factors in this cohort.

LKB1 exonic and whole gene deletions are a common cause of Peutz-Jeghers syndrome.

BACKGROUND: LKB1/STK11 germline mutations cause Peutz-Jeghers syndrome (PJS). The existence of a second PJS locus is controversial, the evidence in its favour being families unlinked to LKB1 and the low frequency of LKB1 mutations found using conventional methods in several studies. Exonic and whole gene deletion or duplication events cannot be detected by routine mutation screening methods. OBJECTIVE: To seek evidence for LKB1 germline deletions or duplications by screening patients meeting clinical criteria for PJS but without detected mutations on conventional screening. METHODS: From an original cohort of 76 patients, 48 were found to have a germline mutation by direct sequencing; the remaining 28 were examined using multiplex ligation dependent probe amplification (MLPA) analysis to detect LKB1 copy number changes. RESULTS: Deletions were found in 11 of the 28 patients (39%)--that is, 14% of all PJS patients (11/76). Five patients had whole gene deletions, two had the promoter and exon 1 deleted, and in one patient exon 8 was deleted. Other deletions events involved: loss of exons 2-10; deletion of the promoter and exons 1-3; and loss of part of the promoter. No duplications were detected. Nine samples with deletions were sequenced at reported single nucleotide polymorphisms to exclude heterozygosity; homozygosity was found in all cases. No MLPA copy number changes were detected in 22 healthy individuals. CONCLUSIONS: These results lessen the possibility of a second PJS locus, as the detection rate of germline mutations in PJS patients was about 80% (59/76). It is suggested that MLPA, or a suitable alternative, should be used for routine genetic testing of PJS patients in clinical practice.

Exon 3 beta-catenin mutations are specifically associated with colorectal carcinomas in hereditary non-polyposis colorectal cancer syndrome.

BACKGROUND AND AIMS: Activating beta-catenin mutations in exon 3 have been implicated in colorectal tumorigenesis. Although reports to the contrary exist, it has been suggested that beta-catenin mutations occur more often in microsatellite unstable (MSI+) colorectal carcinomas, including hereditary non-polyposis colorectal cancer (HNPCC), as a consequence of defective DNA mismatch repair. We have analysed 337 colorectal carcinomas and adenomas, from both sporadic cases and HNPCC families, to provide an accurate assessment of beta-catenin mutation frequency in each tumour type. METHODS: Direct sequencing of exon 3 of beta-catenin. RESULTS: Mutations were rare in sporadic (1/83, 1.2%) and HNPCC adenomas (1/37, 2.7%). Most of the sporadic adenomas analysed (80%) were small (<1 cm), and our data therefore differ from a previous report of a much higher mutation frequency in small adenomas. No oncogenic beta-catenin mutations were identified in 34 MSI+ and 78 microsatellite stable (MSI-) sporadic colorectal cancers but a raised mutation frequency (8/44, 18.2%) was found in HNPCC cancers; this frequency was significantly higher than that in HNPCC adenomas (p=0.035) and in both MSI- (p<0.0001) and MSI+ (p=0.008) sporadic cancers. Mutations were more common in higher stage (Dukes' stages C and D) cancers (p=0.001). CONCLUSION: Exon 3 beta-catenin mutations are associated specifically with malignant colorectal tumours in HNPCC; mutations appear not to result directly from deficient mismatch repair. Our data provide evidence that the genetic pathways of sporadic MSI+ and HNPCC cancers may be divergent, and indicate that mutations in the HNPCC pathway of colorectal tumorigenesis may be determined by selection, not simply by hypermutation.

Evidence for genetic predisposition to desmoid tumours in familial adenomatous polyposis independent of the germline APC mutation.

BACKGROUND: Many patients with familial adenomatous polyposis (FAP) die from desmoid tumours which can arise spontaneously but often appear to be surgically induced by prophylactic colectomy. FAP results from germline adenomatous polyposis coli (APC) gene mutations and desmoids arise following biallelic APC mutation, with one change usually occurring distal to the second beta-catenin binding/degradation repeat of the gene (3' to codon 1399). We have suggested that because families with germline mutations in this region already have the requisite change, they are more likely to develop desmoids. However, there are families with 5' germline mutations where desmoids are common. PATIENTS AND METHODS: We examined desmoid risk dependent on germline APC mutation, sex, history of abdominal surgery, and family history in FAP patients from the St Mark's Hospital Polyposis Registry. RESULTS: Overall desmoid prevalence was 15%. Desmoids tended to cluster in susceptible individuals, irrespective of the germline APC mutation. Independent predictors of increased desmoid risk were: germline mutation distal to codon 1399; any family history of disease; and a strong family history of desmoids. A family history of multiple desmoids (>1) increased an individual's own risk of multiplicity. Females had twice the odds of developing desmoids compared with males. There was no significant interaction between any of the three explanatory variables. CONCLUSIONS: Our results indicate the influence of unknown genetic factors independent of APC in susceptibility to desmoid tumours in FAP. The data have implications in terms of clinical management of FAP patients and assessing the balance between chemoprevention and prophylactic colectomy.

Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line.

PURPOSE: To compare the chromosomal radiosensitivity of C3H mouse acute myeloid leukaemia (AML) cell lines 7926 and 8709 and to investigate the mechanistic basis of the radiosensitivity observed in 7926. MATERIALS AND METHODS: Yields of chromosome aberrations following X-irradiation were determined in Giemsa-stained metaphases. Cell cycle phase distributions were determined by BrdU incorporation and microscopy, apoptosis was assessed by caspase assays. Telomerase activity (TRAP assay), telomere length (Q-FISH and Southern blotting) and telomere function (Robertsonian-like fusion formation) were also examined. The expression levels of telomerase components, telomerase regulators and DNA PKcs were determined on Northern blots. RESULTS: A total of 4.5-7.6-fold elevated chromosome aberration yields were found in 7926 by comparison with 8709 3-24h after 0.5 and 1 Gy X-ray exposure. This difference could not be accounted for by differences in chromatid break-rejoining rates, cell cycle phase distribution or the induction of apoptosis. Telomeres and telomerase were dysfunctional in 7926. However, average telomere length was approximately two-fold greater than in 8709. CONCLUSION: Defective telomere function in 7926 correlates with chromosomal radiosensitivity. This implicates telomere function in addition to telomere length as a determinant of chromosomal radiosensitivity.

Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.

Reports differ as to whether reconstitution of telomerase activity alone is sufficient for immortalization of different types of human somatic cells or whether additional activities encoded by other "immortalizing" genes are also required. Here we show that ectopic expression of either the catalytic subunit of human telomerase (hTERT) or a temperature-sensitive mutant (U19tsA58) of simian virus 40 large-tumor antigen alone was not sufficient for immortalization of freshly isolated normal adult human mammary fibroblasts and endothelial cells. However, a combination of both genes resulted in the efficient generation of immortal cell lines irrespective of the order in which they were introduced or whether they were introduced early or late in the normal proliferative lifespan of the cultures. The order and timing of transduction, however, did influence genomic stability. Karyotype analysis indicated that introduction of both transgenes at early passage, with hTERT first, yielded diploid cell lines. Temperature-shift experiments revealed that maintenance of the immortalized state depended on continued expression of functional U19tsA58 large-tumor antigen, with hTERT alone unable to maintain growth at nonpermissive temperatures for U19tsA58 large-tumor antigen. Such conditional diploid lines may provide a useful resource for both cell engineering and for studies on immortalization and in vitro transformation.

Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription.

X-irradiation of two mouse myeloid leukaemia cell lines was found to lead to increased telomerase activities. Maximal increases in activity at 24 h post-irradiation were approximately three times control unirradiated cell levels. These maxima were reached at between 3-5 Gy depending upon cell line. Peak activity was reached at 8h, remained elevated to 24 h and returned to control levels by 48 h. In contrast, X-irradiation did not activate telomerase in a telomerase-negative human fibroblast line, while in cultured normal mouse bone marrow cells irradiation appeared to reduce activities. No simple relationship between radiation-induced increases in telomerase activity in the myeloid leukaemia lines and the proportions of cells in the S or M phases of the cell cycle was apparent. Radiation-induced increases in activity were significantly reduced by inhibitors of transcription (actinomycin D, alpha-amanatin) and protein synthesis (cycloheximide). These data are consistent with two possibilities: (i) X-irradiation leads to increased transcription and/or translation of a component of telomerase, thus increasing activities; or (ii) X-irradiation induces the transcription of a positive regulator of telomerase activity. Northern blot analysis did not indicate that transcription of mTert, the catalytic subunit of telomerase, or mTerc, the RNA component, was elevated after irradiation. Similarly, no significant changes in the expression of Myc or Tnks, the tankyrase gene, two suspected telomerase regulators, were detected. These data are therefore consistent with the induction by X-irradiation of a positive regulator of telomerase activity other than Tnks or Myc or the core protein and RNA components of the enzyme.

A telomere-independent senescence mechanism is the sole barrier to Syrian hamster cell immortalization.

Reactivation of telomerase and stabilization of telomeres occur simultaneously during human cell immortalization in vitro and the vast majority of human cancers possess high levels of telomerase activity. Telomerase repression in human somatic cells may therefore have evolved as a powerful resistance mechanism against immortalization, clonal evolution and malignant progression. The comparative ease with which rodent cells immortalize in vitro suggests that they have less stringent controls over replicative senescence than human cells. Here, we report that Syrian hamster dermal fibroblasts possess substantial levels of telomerase activity throughout their culture life-span, even after growth arrest in senescence. In our studies, telomerase was also detected in uncultured newborn hamster skin, in several adult tissues, and in cultured fibroblasts induced to enter the post-mitotic state irreversibly by serum withdrawal. Transfection of near-senescent dermal fibroblasts with a selectable plasmid vector expressing the SV40 T-antigen gene resulted in high-frequency single-step immortalization without the crisis typically observed during the immortalization of human cells. Collectively, these data provide an explanation for the increased susceptibility of rodent cells to immortalization (and malignant transformation) compared with their human equivalents, and provide evidence for a novel, growth factor-sensitive, mammalian senescence mechanism unrelated to telomere maintenance.

Role of telomeric sequences in murine radiation-induced myeloid leukaemia.

A previous study indicated that a highly inbred CBA/H mouse colony contained four genotypic variants for telomere-like repeat (TLR) sequence arrays and that one variant subpopulation that constituted 20% of the colony contributed the vast majority (> 90%) of radiation-induced acute myeloid leukaemias (AMLs). Through screening of a satellite CBA/H colony and rescreening of the original colony, we show that, whereas germline telomere sequence polymorphism is frequent in CBA/H mice, there is no genetic link between a specific TLR locus variant and susceptibility to AML. Studies on telomere-hybridising fragments between 200 bp and 150 kb revealed that the germline telomere mutation frequency was highest for restriction fragments > 50 kb. The hypervariability of these high-molecular-weight fragments resulted in each CBA/H mouse from the highly inbred colony having a different genotype. Although it was not possible to ascribe a specific somatic telomere mutation to AML development, telomere rearrangements were common in induced AMLs. Some terminal telomere-hybridising restriction fragments were shortened in AML samples in comparison with normal tissue, but, insofar as the reduction in size was relatively small, it seems unlikely that telomere erosion is a major contributor to the molecular pathology of murine radiation-induced AML.

Microsatellite analysis of recurrent chromosome 2 deletions in acute myeloid leukaemia induced by radiation in F1 hybrid mice.

Deletions and/or rearrangements involving one copy of chromosome 2 are consistent and early events in the development of murine acute myeloid leukaemia (AML) by radiation. More than 90% of AMLs induced in the CBA strain of mice express such cytogenetic alterations, with chromosome 2 breakpoints clustering in the C and F regions of the chromosome. In inbred mouse strains, the molecular resolution of these breakpoints is problematic. However, by using x-ray-induced AMLs in FI progeny of genetically divergent CBA/H x C57BI, it has been possible to show region-specific loss of heterozygosity (LOH) in genetically linked sets of chromosome 2 microsatellite alleles from one of the two parental chromosomes. In the majority of cases, an acceptable concordance was shown for AML chromosome 2 deletion, as defined by microsatellites and as revealed by G-band cytogenetics. A degree of breakpoint clustering was found, but the identification of a number of deletion types, based on the position of proximal and distal breakpoints as defined by microsatellite analysis, strongly supports a leukaemogenic mechanism involving gene deletion. No bias towards loss of CBA or C57BI alleles was observed, and the gender of AML-presenting animals did not appear to influence the parental origin of the deletions. A molecular map of chromosome 2 breakpoints has now been established in FI AMLs as a first step towards the molecular cloning of breakpoint sequences.

The IL-1 alpha and beta genes are closely linked (less than 70 kb) on mouse chromosome 2.

The murine IL-1 alpha and IL-1 beta genes encode structurally and evolutionarily related cytokines that exert a regulatory role in numerous physiological processes including hemopoiesis. Previous studies have shown these genes to be closely linked in the F region of mouse chromosome 2. Here we show, using pulsed-field gel electrophoresis, that the IL-1 alpha and beta genes of the CBA/H mouse are very closely linked and contained within a SmaI genomic fragment of approximately 70 kb. From conventional and PFGE analyses we suggest that IL-1 beta lies 5' to IL-1 alpha and that the two genes are in the same orientation and separated by approximately 50 kb. The apparent clustering of such hemopoietic genes is discussed in relation to evolutionary tandem gene duplication and possible associations with chromosomal fragile sites and leukemogenesis.

Preliminary molecular studies on two chromosome 2 encoded genes, c-abl and beta 2M, in radiation-induced murine myeloid leukaemias.

The majority of radiation-induced murine myeloid leukaemias are characterized by deletion and/or translocation of an interstitial region of chromosome 2, and there is evidence that such events may occur very early in myeloid leukaemogenesis. Analyses presented and discussed here on the structure and function of two possibly relevant chromosome 2 encoded genes (c-abl and beta 2M) lead to the preliminary conclusion that neither are directly involved nor activationally changed by the characteristic chromosome 2 rearrangements.

Mechanism of action of some bioreducible 2-nitroimidazoles: comparison of in vitro cytotoxicity and ability to induce DNA strand breakage.

The interaction of a series of nitroimidazole-aziridine radiosensitisers, as parent or radiation-reduced species, with plasmid DNA in aqueous solution at pH7 results in strand breakage. The efficiency of strand breakage substantially increases on reduction of the nitroimidazole analogues. The rate of production of strand breaks decreases on interaction with both parent and reduced nitroimidazole analogues as the aziridine moiety is deactivated through alkyl-substitution. These variations in efficiency are reflected in changes in the toxicity towards both oxic and hypoxic cells and in the decrease in toxicity with progressive substitution of the aziridine moiety. The stabilities of these nitroimidazoles in aqueous solution at pH7 have also been determined. However, these stabilities do not parallel the variations in the alkylating efficiency of DNA by the aziridine moiety. These results have been discussed in terms of the relative reactivities of the nitroimidazoles with plasmid DNA and their ability to act as cytotoxic agents, especially following bioreduction and how the findings may relate to the radiosensitising properties of these agents.

Induction of DNA strand breaks by reduced nitroimidazoles. Implications for DNA base damage.

Radiation-reduced 2-nitroimidazoles (misonidazole, RSU-1137, Ro.03-8799 and Ro.03-8800) incubated in air with plasmid DNA (pH 7.0, 310K) induce DNA strand breakage, as revealed following subsequent heat or alkali treatment. Only RSU-1137 resulted in the binding of a [2-14C] fragment and significant yields of heat-labile strand breaks (greater than 20% loss of type-I DNA after 48 hr incubation). RSU-1137 was shown to be greater than 6 times more effective than misonidazole at producing alkali-labile breaks. In fact, the efficiency of alkali-induced strand break production is in the order: misonidazole less than Ro.03-8799 approximately Ro.03-8800 less than RSU-1137. Reaction of these reduced 2-nitroimidazoles with 2'-deoxyguanosine (dG) also results in the formation of a common glyoxal-dG product, with its yield and rate of production being dependent upon the 2-nitroimidazole used. It has been demonstrated that these variations are influenced by the N-1 side-chain of the 2-nitroimidazole. Product yields are approximately 5-6 times greater with misonidazole than with RSU-1137. From the evidence presented, it is apparent that formation of glyoxal (or a glyoxal-like product) is not responsible for the DNA strand breakage seen. It is inferred that these breaks are induced by a nitro-reduction product(s) which remains unidentified.

Studies on the mechanisms of the radiosensitizing and cytotoxic properties of RSU-1069 and its analogues.

RSU 1069 is a substantially more efficient sensitizer than misonidazole when hypoxic Chinese hamster V79 cells are irradiated in vitro at room temperature; such that for 0.5 mmol dm-3 sensitizer an ER of 3.0 is obtained for RSU 1069 whereas an ER of only 1.6 is obtained for misonidazole. However, when irradiation is done at 4 degrees C, the radiosensitization caused by RSU 1069 is reduced to a level close to that obtained with misonidazole, the action remaining unaltered at the lower temperature. This temperature dependent component of sensitization for RSU 1069 suggests the involvement of a slow biochemical process that has an appreciable activation energy. The RSU 1069 analogue RB 7040 is a more efficient radiosensitizer than RSU 1069 particularly at lower concentrations. This compound has a pKa value of 8.45, in contrast to that of RSU 1069 which is 6.04. Weak bases with pKa values in excess of average intra-cellular pH can be taken up preferentially into cells from medium at pH 7.4. It is shown that RB 7040 has a 4 X higher intracellular concentration than RSU 1069 for a similar extracellular concentration. This will explain, at least in part, the greater sensitizing efficiency of this compound when compared to RSU 1069 in vitro.

The phosphate-group of DNA as a potential target for RSU-1069, a nitroimidazole-aziridine radiosensitizer.

Strand breakage of plasmid DNA by parent and radiation-reduced RSU-1069 (2.0-8.0 mmol dm-3) has been measured in air over 4 hr at 310K. Reduced RSU-1069 was shown to be approximately 4 times as efficient as the parent compound at causing strand breakage. The aziridine moiety of both parent and reduced RSU-1069 is required for strand break production and, furthermore, is capable of alkylating inorganic phosphate (k = 1.0 X 10(-3) dm3 mol-1 s-1) and a series of nucleotides (k = 0.8 - 2.1 X 10(-3) dm3 mol-1 s-1) at pH 7.0. From the determined rate constants and the nature of the adducts observed, it was shown that phosphate is a target on nucleotides, although additional sites probably exist particularly, on dGMP and dAMP. The mechanism of action of RSU-1069 is discussed in terms of its ability to act as a cytotoxic agent, radiosensitizer and bioreductive drug.