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Background Cancer stem cells (CSCs) share a number of properties with somatic stem cells including heightened protective mechanisms and the ability to self-renew. CSCs are a critical subpopulation of cancer cells implicated in tumor formation, metastases and recurrence. Methods We used serial colonosphere culture to enrich for CSCs from two human CRC cell lines. The expression of proposed colorectal CSC markers and multi-drug resistance genes were assessed via flow cytometry and RT-qPCR. Drug resistance gene expression and self-renewal ability were also determined following treatment with the chemotherapeutic 5-fluorouracil. Results Colonosphere culture successfully enriched for a subpopulation of cells with CSC-related gene expression and heightened self-renewal ability, particularly in the SW480 cell line. Chemotherapy treatment significantly reduced sphere formation however a small fraction of cells survived treatment and retained their self-renewal ability. Conclusions Our findings support the use of the sphere formation assay to study CSCs. The ability of cells to self-renew following chemotherapy treatment highlights the importance of targeting both the bulk of tumor cells and the CSC population to prevent recurrence in colorectal cancer.

Angiogenesis is essential for mammalian development and tissue homeostasis, and is involved in several pathological processes, including tumor growth and dissemination. Many factors within the tissue microenvironment are known to modulate angiogenesis, including cytokines, such as transforming growth factor beta (TGFβ), and oxygen level. TGFβ exists in three different isoforms (1, 2 and 3), all of which (albeit in different contexts) might mediate angiogenesis and are able to induce endothelial-mesenchymal transition (EndoMT), a process involved in heart development, pathologic fibrosis and, as recently reported, in angiogenesis. Low oxygen level, referred to as hypoxia, has been independently shown to induce angiogenesis, modulate TGFβ signalling and promote EndoMT. However, how these phenomena might be interconnected to drive angiogenesis is rather unexplored. To begin addressing the potential contribution of TGFβ-induced EndoMT to angiogenesis, and to explore how microenvironmental hypoxia might influence these processes, we investigated the effect of TGFβ isoforms 1 and 2 on early EndoMT response in cultured adult endothelium under standard (21 %) and hypoxic (1 %) culture conditions. Our data indicates that EndoMT-like changes, such as an increase in expression and nuclear translocation of Snail, Slug and Zeb1, and reduction of VE-cadherin expression, occur in response to TGFβ1 and/or TGFβ2 as early as 6h after stimulation and might be enhanced by hypoxia in an isoform-specific manner. Further, hypoxia enhances canonical TGFβ signalling, and appears to be a key determinant of Snail's differential involvement in endothelial cell responses to TGFβ1 versus TGFβ2.

Dichloroacetate (DCA) is a metabolic reprogramming agent that reverses the Warburg effect, causing cancer cells to couple glycolysis to oxidative phosphorylation. This has been shown to induce apoptosis and reduce the growth of various types of cancer but not normal cells. Colorectal cancer cells HCT116, HCT116 p53(-/-), and HCT116 Bax(-/-), were treated with DCA in vitro. Response to treatment was determined by measuring PDH phosphorylation, apoptosis, proliferation, and cell cycle. Molecular changes associated with these responses were determined using western immunoblotting and quantitative PCR. Treatment with 20 mM DCA did not increase apoptosis, despite decreasing levels of anti-apoptotic protein Mcl-1 after 6 h, in any of the cell lines observed. Mcl-1 expression was stabilized with MG-132, an inhibitor of proteasomal degradation. A decrease in Mcl-1 correlated with a decrease in proliferation, both of which showed dose-dependence in DCA treated cells. Cells showed nuclear localization of Mcl-1, however cell cycle was unaffected by DCA treatment. These data suggest that a reduction in the prosurvival Bcl-2 family member Mcl-1 due to increased proteasomal degradation is correlated with the ability of DCA to reduce proliferation of HCT116 human colorectal cancer cells without causing apoptosis.

Heat shock proteins (HSPs) are molecular chaperones subdivided into several families based on their molecular weight. Due to their cytoprotective roles, these proteins may help protect cancer cells against chemotherapy-induced cell death. Investigation into the biologic activity of HSPs in a variety of cancers including primary bone tumors, such as osteosarcoma (OSA), is of great interest. Both human and canine OSA tumor samples have aberrant production of HSP70. This study assessed the response of canine OSA cells to inhibition of HSP70 and GRP78 by the ATP-mimetic VER-155008 and whether this treatment strategy could sensitize cells to doxorubicin chemotherapy. Single-agent VER-155008 treatment decreased cellular viability and clonogenic survival and increased apoptosis in canine OSA cell lines. However, combination schedules with doxorubicin after pretreatment with VER-155008 did not improve inhibition of cellular viability, apoptosis, or clonogenic survival. Treatment with VER-155008 prior to chemotherapy resulted in an upregulation of target proteins HSP70 and GRP78 in addition to the co-chaperone proteins Herp, C/EBP homologous transcription protein (CHOP), and BAG-1. The increased GRP78 was more cytoplasmic in location compared to untreated cells. Single-agent treatment also revealed a dose-dependent reduction in activated and total Akt. Based on these results, targeting GRP78 and HSP70 may have biologic activity in canine osteosarcoma. Further studies are required to determine if and how this strategy may impact the response of osteosarcoma cells to chemotherapy.

Background Radiation therapy is a palliative treatment modality for canine osteosarcoma, with transient improvement in analgesia observed in many cases. However there is room for improvement in outcome for these patients. It is possible that the addition of sensitizing agents may increase tumor response to radiation therapy and prolong quality of life. Epidermal growth factor receptor (EGFR) expression has been documented in canine osteosarcoma and higher EGFR levels have been correlated to a worse prognosis. However, effects of EGFR inhibition on radiation responsiveness in canine osteosarcoma have not been previously characterized. This study examined the effects of the small molecule EGFR inhibitor erlotinib on canine osteosarcoma radiation responses, target and downstream protein expression in vitro. Additionally, to assess the potential impact of treatment on tumor angiogenesis, vascular endothelial growth factor (VEGF) levels in conditioned media were measured. Results Erlotinib as a single agent reduced clonogenic survival in two canine osteosarcoma cell lines and enhanced the impact of radiation in one out of three cell lines investigated. In cell viability assays, erlotinib enhanced radiation effects and demonstrated single agent effects. Erlotinib did not alter total levels of EGFR, nor inhibit downstream protein kinase B (PKB/Akt) activation. On the contrary, erlotinib treatment increased phosphorylated Akt in these osteosarcoma cell lines. VEGF levels in conditioned media increased after erlotinib treatment as a single agent and in combination with radiation in two out of three cell lines investigated. However, VEGF levels decreased with erlotinib treatment in the third cell line. Conclusions Erlotinib treatment promoted modest enhancement of radiation effects in canine osteosarcoma cells, and possessed activity as a single agent in some cell lines, indicating a potential role for EGFR inhibition in the treatment of a subset of osteosarcoma patients. The relative radioresistance of osteosarcoma cells does not appear to be related to EGFR signalling exclusively. Angiogenic responses to radiation and kinase inhibitors are similarly likely to be multifactorial and require further investigation.

Pancreatic exocrine and bone marrow dysfunctions are considered to be universal features of Shwachman-Diamond syndrome (SDS) whereas the associated skeletal dysplasia is variable and not consistently observed. The genetic defect in SDS has recently been identified; causative mutations have been shown in the SBDS gene. The aims of this study were to characterize the nature, frequency, and age-related changes of radiographic skeletal abnormalities in patients with SBDS mutations and to assess genotype-phenotype correlation. Fifteen patients (mean age 9.7 years) with a clinical diagnosis of SDS and documented SBDS gene mutations were included. Review of their skeletal radiographs showed abnormalities in all patients. The skeletal changes were variable, even in patients with identical genotypes. The typical features were (1) delayed appearance of secondary ossification centers, (2) variable widening and irregularity of the metaphyses in early childhood, followed by progressive thickening and irregularity of the growth plates, and (3) generalized osteopenia. There was a tendency towards normalization of the epiphyseal maturation defect and progression of the metaphyseal changes with age. The results suggest that the characteristic skeletal changes are present in all patients with SDS and SBDS mutations, but their severity and localization varies with age. No phenotype-genotype correlation was observed.

To evaluate the role of serum enzymes for defining the pancreatic phenotype in Shwachman-Diamond syndrome (SDS), an inherited multisystem condition.Serum pancreatic trypsinogen and isoamylase were measured in 164 patients known or presumed to have SDS. The diagnosis was confirmed in 90 patients. Among 74 unconfirmed cases, 35 ("probable SDS") had hematologic dysfunction but lacked documented pancreatic dysfunction, whereas 39 patients ("improbable SDS") lacked both documented pancreatic and hematologic dysfunction. Classification and regression tree (CART) analysis was performed in 90 patients with SDS and 134 control patients to establish a rule for defining the pancreatic phenotype of SDS; the rule was then applied to the patients with unconfirmed diagnosis.In the control patients, serum trypsinogen showed little variation with age, whereas serum isoamylase values rose from birth on, attaining adult values by 3 years. For patients with SDS, serum trypsinogen values were low in young patients and tended to increase with age, whereas serum isoamylase values remained low at all ages. The CART rule combined results from both enzymes and classified the pancreatic phenotype in all but one SDS patient, who was3 years of age. Excluding patients3 years of age, CART identified the pancreatic phenotype in 82% and 7% of the "probable SDS" and "improbable SDS" cases, respectively.Serum pancreatic enzymes are useful for determining the pancreatic phenotype and confirming the diagnosis of SDS.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. A genomewide scan of families with SDS was terminated at approximately 50% completion, with the identification of chromosome 7 markers that showed linkage with the disease. Finer mapping revealed significant linkage across a broad interval that included the centromere. The maximum two-point LOD score was 8.7, with D7S473, at a recombination fraction of 0. The maximum multipoint LOD score was 10, in the interval between D7S499 and D7S482 (5.4 cM on the female map and 0 cM on the male map), a region delimited by recombinant events detected in affected children. Evidence from all 15 of the multiplex families analyzed provided support for the linkage, consistent with a single locus for SDS. However, the presence of several different mutations is suggested by the heterogeneity of disease-associated haplotypes in the candidate region.

Shwachman-Diamond syndrome is a rare genetic disorder of unknown pathogenesis involving exocrine pancreatic insufficiency and hematological and skeletal abnormalities. There is broad clinical variability; the extent of heterogeneity is unknown but comparisons within a large cohort of patients show no striking differences between patients of families with single or multiple affected offspring. Segregation analysis of a cohort of 69 families has suggested an autosomal recessive mode of inheritance. A single constitutional de novo chromosome rearrangement was reported in a Japanese patient involving a balanced translocation, t(6;12)(q16.2;q21.2), thereby suggesting possible loci for a genetic defect. Evenly spaced microsatellite markers spanning 26-32 cM intervals from D6S1056 to D6S304 and D12S375 to D12S346 were analyzed for linkage in members of 13 Shwachman-Diamond syndrome families with two or three affected children. Two-point lod scores were calculated for each marker under assumptions of recessive inheritance and complete penetrance. Negative lod scores indicated exclusion of both chromosome regions. Further, affected sibs were discordant for inheritance of chromosomes in most families based on constructed haplotypes. The cytogenetic abnormality is not associated with most cases of Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS; OMIM 260400) is an autosomal recessive disorder with clinical features that include pancreatic exocrine insufficiency, hematological dysfunction and skeletal abnormalities. Here, we report identification of disease-associated mutations in an uncharacterized gene, SBDS, in the interval of 1.9 cM at 7q11 previously shown to be associated with the disease. We report that SBDS has a 1.6-kb transcript and encodes a predicted protein of 250 amino acids. A pseudogene copy (SBDSP) with 97% nucleotide sequence identity resides in a locally duplicated genomic segment of 305 kb. We found recurring mutations resulting from gene conversion in 89% of unrelated individuals with SDS (141 of 158), with 60% (95 of 158) carrying two converted alleles. Converted segments consistently included at least one of two pseudogene-like sequence changes that result in protein truncation. SDBS is a member of a highly conserved protein family of unknown function with putative orthologs in diverse species including archaea and eukaryotes. Archaeal orthologs are located within highly conserved operons that include homologs of RNA-processing genes, suggesting that SDS may be caused by a deficiency in an aspect of RNA metabolism that is essential for development of the exocrine pancreas, hematopoiesis and chrondrogenesis.

Objectives: With the use of clinical data from a large international cohort, we evaluated and compared affected siblings and isolated cases. Study design: Data from 116 families were collected, and patients conforming to our predetermined diagnostic criteria were analyzed. Phenotypic manifestations of affected siblings and singletons were compared with the use of t tests, Wilcoxon scores, and χ2 analysis. Results: Eighty-eight patients (33 female, 55 male; median age 5.20 years) fulfilled our predetermined diagnostic criteria for Shwachman syndrome; 63 patients were isolated cases, and 25 affected siblings were from 12 multiplex families. Steatorrhea was present in 86% (57 of 66), and 91% (78 of 86) displayed a low serum trypsinogen concentration. Patients older than 4 years more often had pancreatic sufficiency. Neutropenia occurred in 98%, anemia in 42%, and thrombocytopenia in 34%. Myelodysplasia or cytogenetic abnormalities were reported in 7 patients. Short stature with normal nutritional status was a prominent feature. Conclusions: Clinical features among patients with Shwachman syndrome varied between patients and with age. Similarities in phenotype between isolated cases and affected sibling sets support the hypothesis that Shwachman syndrome is a single disease entity. (J Pediatr 1999;135:81-8.)

Shwachman-Diamond syndrome is a rare disorder of unknown cause. Reports have indicated the occurrence of affected siblings, but formal segregation analysis has not been performed. In families collected for genetic studies, the mean paternal age and mean difference in parental ages were found to be consistent with the general population. We determined estimates of segregation proportion in a cohort of 84 patients with complete sibship data under the assumption of complete ascertainment, using the Li and Mantel estimator, and of single ascertainment with the Davie modification. A third estimate was also computed with the expectation-maximization (EM) algorithm. All three estimates supported an autosomal recessive mode of inheritance, but complete ascertainment was found to be unlikely. Although there are no overt signs of disease in adult carriers (parents), the use of serum trypsinogen levels to indicate exocrine pancreatic dysfunction was evaluated as a potential measure for heterozygote expression. No consistent differences were found in levels between parents and a normal control population. Although genetic heterogeneity cannot be excluded, our results indicate that simulation and genetic analyses of Shwachman-Diamond syndrome should consider a recessive model of inheritance.