Search

The presence of tumor immune infiltrating cells (TILs), particularly CD8+ T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8+ T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8+ and CD45RO+ -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8+ TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8+ TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.

The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation.

Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb+/− mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers–Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfβ−1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.

Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.

The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5(+) stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5(+) stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5(+) intestinal stem cells.

BACKGROUND: Conventional laparoscopic surgery uses CO2 that is dry and cold, which can damage peritoneal surfaces. It is speculated that disseminated cancer cells may adhere to such damaged peritoneum and metastasize. We hypothesized that insufflation using humidified-warm CO2, which has been shown to reduce mesothelial damage, will also ameliorate peritoneal inflammation and tumor cell implantation compared to conventional dry-cold CO2. METHODS: Laparoscopic insufflation was modeled in mice along with anesthesia and ventilation. Entry and exit ports were introduced to maintain insufflation using dry-cold or humidified-warm CO2 with a constant flow and pressure for 1 h; then 1000 or 1 million fluorescent-tagged murine colorectal cancer cells (CT26) were delivered into the peritoneal cavity. The peritoneum was collected at intervals up to 10 days after the procedure to measure inflammation, mesothelial damage, and tumor burden using fluorescent detection, immunohistochemistry, and scanning electron microscopy. RESULTS: Rapid temperature control was achieved only in the humidified-warm group. Port-site tumors were present in all mice. At 10 days, significantly fewer tumors on the peritoneum were counted in mice insufflated with humidified-warm compared to dry-cold CO2 (p < 0.03). The inflammatory marker COX-2 was significantly increased in the dry-cold compared to the humidified-warm cohort (p < 0.01), while VEGFA expression was suppressed only in the humidified-warm cohort. Significantly less mesothelial damage and tumor cell implantation was evident from 2 h after the procedure in the humidified-warm cohort. CONCLUSIONS: Mesothelial cell damage and inflammation are reduced by using humidified-warm CO2 for laparoscopic oncologic surgery and may translate to reduce patients' risk of developing peritoneal metastasis.

MYB transcriptional elongation is regulated by an attenuator sequence within intron 1 that has been proposed to encode a RNA stem loop (SLR) followed by a polyU tract. We report that NFekBp50 can bind the SLR polyU RNA and promote MYB transcriptional elongation together with NFekBp65. We identified a conserved lysine-rich motif within the Rel homology domain (RHD) of NFekBp50, mutation of which abrogated the interaction of NFekBp50 with the SLR polyU and impaired NFekBp50 mediated MYB elongation. We observed that the TAR RNA-binding region of Tat is homologous to the NFekBp50 RHD lysine-rich motif, a finding consistent with HIV Tat acting as an effector of MYB transcriptional elongation in an SLR dependent manner. Furthermore, we identify the DNA binding activity of NFekBp50 as a key component required for the SLR polyU mediated regulation of MYB. Collectively these results suggest that the MYB SLR polyU provides a platform for proteins to regulate MYB and reveals novel nucleic acid binding properties of NFekBp50 required for MYB regulation.

© 2015 The Authors. The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5+ stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5+ stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5+ intestinal stem cells.

The cyclic-AMP response element binding (CREB) protein has been shown to have a pivotal role in cell survival and cell proliferation. Transgenic rodent models have revealed a role for CREB in higher-order brain functions, such as memory and drug addiction behaviors. CREB overexpression in transgenic animals imparts oncogenic properties on cells in various tissues, and aberrant CREB expression is associated with tumours. It is the central position of CREB, downstream from key developmental and growth signalling pathways, which gives CREB this ability to influence a spectrum of cellular activities, such as cell survival, growth and differentiation, in both normal and cancer cells. We show that CREB is highly expressed and constitutively activated in patient glioma tissue and that this activation closely correlates with tumour grade. The mechanism by which CREB regulates glioblastoma (GBM) tumour cell proliferation involves activities downstream from both the mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K) pathways that then modulate the expression of three key cell cycle factors, cyclin B, D and proliferating cell nuclear antigen (PCNA). Cyclin D1 is highly CREB-dependent, whereas cyclin B1 and PCNA are co-regulated by both CREB-dependent and -independent mechanisms. The precise regulatory network involved appears to differ depending on the tumour-suppressor phosphatase and tensin homolog status of the GBM cells, which in turn allows CREB to regulate the activity of the PI3K itself. Given that CREB sits at the hub of key cancer cell signalling pathways, understanding the role of glioma-specific CREB function may lead to improved novel combinatorial anti-tumour therapies, which can complement existing PI3K-specific drugs undergoing early phase clinical trials.

Copyright © 2014 American Association for the Advancement of Science. All Rights Reserved. Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt-to-ß-catenin pathway in the intestinal epithelium. Because Wnt-ß-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt-ß-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.

The gastrointestinal (GI) epithelium is constantly renewing, depending upon the intestinal stem cells (ISC) regulated by a spectrum of transcription factors (TFs), including Myb. We noted previously in mice with a p300 mutation (plt6) within the Myb-interaction-domain phenocopied Myb hypomorphic mutant mice with regard to thrombopoiesis, and here, changes in GI homeostasis. p300 is a transcriptional coactivator for many TFs, most prominently cyclic-AMP response element-binding protein (CREB), and also Myb. Studies have highlighted the importance of CREB in proliferation and radiosensitivity, but not in the GI. This prompted us to directly investigate the p300-Myb-CREB axis in the GI. Here, the role of CREB has been defined by generating GI-specific inducible creb knockout (KO) mice. KO mice show efficient and specific deletion of CREB, with no evident compensation by CREM and ATF1. Despite complete KO, only modest effects on proliferation, radiosensitivity and differentiation in the GI under homeostatic or stress conditions were evident, even though CREB target gene pcna (proliferating cell nuclear antigen) was downregulated. creb and p300 mutant lines show increased goblet cells, whereas a reduction in enteroendocrine cells was apparent only in the p300 line, further resembling the Myb hypomorphs. When propagated in vitro, crebKO ISC were defective in organoid formation, suggesting that the GI stroma compensates for CREB loss in vivo, unlike in MybKO studies. Thus, it appears that p300 regulates GI differentiation primarily through Myb, rather than CREB. Finally, active pCREB is elevated in colorectal cancer (CRC) cells and adenomas, and is required for the expression of drug transporter, MRP2, associated with resistance to Oxaliplatin as well as several chromatin cohesion protein that are relevant to CRC therapy. These data raise the prospect that CREB may have a role in GI malignancy as it does in other cancer types, but unlike Myb, is not critical for GI homeost

Gastrointestinal (GI) homeostasis requires the action of multiple pathways. There is some controversy regarding whether small intestine (SI) Paneth cells (PCs) play a central role in orchestrating crypt architecture and their relationship with Lgr5+ve stem cells. Nevertheless, we previously showed that germline CSF-1 receptor (Csf1r) knock out (KO) or Csf1 mutation is associated with an absence of mature PC, reduced crypt proliferation and lowered stem cell gene, Lgr5 expression. Here we show the additional loss of CD24, Bmi1 and Olfm4 expression in the KO crypts and a high resolution 3D localization of CSF-1R mainly to PC. The induction of GI-specific Csf1r deletion in young adult mice also led to PC loss over a period of weeks, in accord with the anticipated long life span of PC, changed distribution of proliferating cells and this was with a commensurate loss of Lgr5 and other stem cell marker gene expression. By culturing SI organoids, we further show that the Csf1r(-/-) defect in PC production is intrinsic to epithelial cells as well as definitively affecting stem cell activity. These results show that CSF-1R directly supports PC maturation and that in turn PCs fashion the intestinal stem cell niche.

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1(op/op) mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r(-/-) and Csf1(op/op) mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r(-/-) colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r(+/-) male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r(+/-) female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.

Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67-negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. ©2011 AACR.

Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi-1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi-1 is induced with IR to half the level in mutant mice. dcamkl-1 and olfm4 failed to recover after IR in both wild-type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/plt4 mice. Self-renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell-containing tissues. © AlphaMed Press.

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were indentified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/2animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/2animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have i

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were identified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/- animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/- animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues.

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were indentified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21(+/-) animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21(+/-) animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues.

The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation., The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation.