Telomere dysfunction has been associated with chromosomal instability in colorectal carcinoma, but the consequences of telomere-dependent instability for chromosome integrity and clonal evolution have been little explored. We show here that abnormally short telomeres lead to a wide spectrum of mitotic disturbances in colorectal cancer cell lines, including anaphase bridging, wholechromosome lagging, and mitotic multipolarity. These abnormalities were found in both the presence and absence of microsatellite instability. The mean telomere length varied extensively between cells from the same tumor, allowing the establishment of tumor cell subpopulations with highly different frequencies of mitotic disturbances. Anaphase bridging typically resulted in either intercentromeric chromatin fragmentation or centromere detachment, leading to pericentromeric chromosome rearrangements and loss of whole chromosomes, respectively. There was a strong correlation between anaphase bridges and multipolar mitoses, and the induction of dicentric chromosomes by gamma irradiation and telomerase inhibition led to an elevated frequency of multipolar mitotic spindles, suggesting that multipolarity could result from polyploidization triggered by anaphase bridging. Chromatid segregation in multipolar mitoses was close to random, resulting in frequent nullisomies and nonviable daughter cells. In contrast, there was a high clonogenic survival among cells having gone through anaphase bridging in bipolar mitoses. Bridging of telomere-deficient chromosomes could thus be a major mutational mechanism in colorectal cancer, whereas mitotic multipolarity appears to be a secondary phenomenon that rarely, if ever, contributes to clonal evolution. chromosome instability ͉ telomere dysfunction ͉ colorectal cancer ͉ cytogenetics ͉ telomerase T wo major modes of mutation have been demonstrated in colorectal carcinomas, microsatellite instability (MIN) and chromosomal instability (CIN). MIN is typically caused by mutations in mismatch repair genes, whereas several different molecular mechanisms have been associated with CIN, including mutations in mitotic checkpoint genes (1, 2), mutation and͞or loss of the APC gene (3, 4), microtubule spindle defects (4), and viral infection (5). Also telomere dysfunction has been implicated in colorectal carcinogenesis and CIN (6, 7). Transgenic APC Min Terc Ϫ/Ϫ mice with short telomeres show an increased rate of colorectal adenoma initiation (8), and significant telomere shortening has been detected at the earliest stages of invasive colorectal cancer in human material (9). However, the precise consequences of telomere shortening for chromosomal integrity remain unclear. The present study demonstrates that telomere dysfunction does not lead to chromosome aberrations through a single mechanism. Instead, it causes a spectrum of mitotic defects with anaphase bridges triggering both numerical and structural chromosome changes, and with mitotic multipolarity leading to more dramatic genomic changes that may prove deleteriou...

Enforced expression of IntroductionHematopoiesis relies on the unique abilities of relatively few hematopoietic stem cells to self-renew and generate progenitors that will differentiate into the mature cells forming the blood system. This dynamic process is tightly regulated by a complex of internal and external signals, such as transcription factors, growth factors, and cell cycle regulators (for reviews, see Orkin 1 and Verfaillie 2 ). Many transcription factors, including homeobox (Hox) transcription factors, have been shown to be key players in the proliferation and differentiation of early progenitor cells. 3,[4][5][6] Specific expression patterns of multiple Hox genes have been detected in normal and leukemic hematopoiesis. 7,8 Enforced expression of Hox genes has been shown to affect the ability of progenitors and stem cells to proliferate and differentiate. [9][10][11][12][13][14][15][16][17] One of these genes, Hoxb4, has been implicated in the regulation of hematopoietic stem cell regeneration, 8 and retrovirally engineered overexpression in murine bone marrow cells dramatically increases the stem cell pool ex vivo and in vivo, resulting in faster, more complete recovery of the stem cells in transplantation studies with no adverse effect on differentiation or lineage distribution. 14,[18][19][20][21] This is in contrast to the overexpression of other Hox genes, which can perturb the proliferation and lineage commitment of primitive progenitors and can give rise to hematopoietic malignancies. 10,11,13,15,16,[22][23][24] However, recent studies have suggested that the effect of Hoxb4 is concentration dependent and is not necessarily restricted to proliferation. [25][26][27] Thus, the level of Hoxb4 expression has to be within a specific range for Hoxb4 to increase stem cell proliferation without adverse effects on differentiation. Although enforced expression of Hoxb4 in hematopoietic cells has been studied in detail, its physiologic role in hematopoiesis is poorly understood. Recently, we described a mouse model deficient in Hoxb3 and Hoxb4, showing reduced proliferative capacity of the stem cell pool without otherwise perturbing hematopoiesis. 28 Here we report a novel mouse model in which the Hoxb4 gene alone has been completely removed through the Cre/loxP technique. Hoxb4-deficient mice have a phenotype similar to that of double Hoxb3/Hoxb4 knockout (KO) mice, although the effects are milder in the Hoxb4 Ϫ/Ϫ mice. The phenotype observed seems mainly confined to the stem cell pool, resulting in reduced proliferative capacity of bone marrow and fetal liver hematopoietic stem cells (HSCs) without affecting differentiation or lineage choice. Deficiency of Hoxb4 or Hoxb3 and Hoxb4 affects the expression of other Hox genes and the expression of cell cycle regulators, indicating a complex regulatory role of these Hox genes. Collectively, these findings indicate that Hoxb4 improves proliferative recruitment of HSCs in settings demanding high proliferation, such as transplantation, but that it has a less pro...

Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin ؊ ScaI ؉ c-kit ؉ hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response.Class I Homeobox (Hox) genes encode a family of 39 transcription factors sharing a highly conserved DNA-binding domain. In mammals they play a major role in specifying position and tissue fate in the embryo, as has been demonstrated by several lack-of-function Hox gene mutants that exhibit various developmental abnormalities (see, for example, references 6, 29, 30, 38, 41, 49, and 58). Hox genes are also expressed postnatally, and several of them are expressed in primitive hematopoietic cells and committed progenitors but downregulated upon differentiation to mature cells (44).Murine models have been generated where enforced expression of Hox genes is used to determine the effect of overexpression on self-renewal, differentiation, and other cell fate decisions during hematopoiesis (for reviews, see references 10 and 55). Such models include overexpression of HOXA10, as well as HOXA9, which both affected myelo-and lymphopoiesis and ultimately lead to myeloid leukemia (5, 9, 23, 52, 53). Expression of HOXB3 and HOXB4 is found in the primitive CD34 ϩ population that is highly enriched for human hematopoietic stem cells (HSCs) but is rapidly downregulated as the cells differentiate into committed progenitors (44). Despite very similar expression pattern of HOXB3 and HOXB4, suggesting a common role or collaboration between these factors, the consequences from overexpressing these genes are very different. Although enforced expression of HOXB3 blocks both T-and B-cell development and causes a myeloproliferative disorder (46), overexpression of HOXB4 greatly...

Background: The cell surface glycoprotein E-cadherin (CDH1) is a key regulator of adhesive properties in epithelial cells. Germline mutations in CDH1 are well established as the defects underlying hereditary diffuse gastric cancer (HDGC) syndrome, and an increased risk of lobular breast cancer (LBC) has been described in HDGC kindreds. However, germline CDH1 mutations have not been described in patients with LBC in non-HDGC families. This study aimed to investigate the frequency of germline CDH1 mutations in patients with LBC with early onset disease or family histories of breast cancer without DGC. Methods: Germline DNA was analysed in 23 women with invasive lobular or mixed ductal and lobular breast cancers who had at least one close relative with breast cancer or had themselves been diagnosed before the age of 45 years, had tested negative for a germline BRCA1 or BRCA2 mutation, and reported no personal or family history of diffuse gastric cancer. The full coding sequence of CDH1 including splice junctions was amplified using PCR and screened for mutations using DHPLC and sequencing. Results: A novel germline CDH1 truncating mutation in the extracellular portion of the protein (517insA) was identified in one woman who had LBC at the age of 42 years and a first degree relative with invasive LBC. Conclusions: Germline CDH1 mutations can be associated with invasive LBC in the absence of diffuse gastric cancer. The finding, if confirmed, may have implications for management of individuals at risk for this breast cancer subtype. Clarification of the cancer risks in the syndrome is essential.