c-Jun NH2-terminal kinase 1 plays a critical role in intestinal homeostasis and tumor suppression

The c-Jun NH(2)-terminal kinase (JNK) signal transduction pathway plays important roles in cellular processes and stress. However, the role of JNK1 in intestinal homeostasis and tumorigenesis is unknown. Therefore, we used a JNK1 knockout mouse model to characterize intestinal cell maturation and tumorigenesis. In addition, colon cancer cell lines were used to validate the role of JNK1 and to elucidate the underlying molecular mechanisms in vitro. To our surprise, we found that mice with targeted inactivation of JNK1 spontaneously developed intestinal tumors. The normal mucosa in JNK1-deficient mice showed decreased cell differentiation and increased cell proliferation. This tumorigenesis was closely linked to the down-regulation of p21(WAF1/cip1), a cyclin-dependent kinase inhibitor, in intestinal epithelial cells. Immunohistochemical staining showed that JNK1 was highly expressed in the differentiation compartment of the intestinal mucosa and that the expression of JNK1 was significantly decreased in both human colonic and mouse intestinal tumors. In the colon cancer cell lines, JNK1 expression was up-regulated during spontaneous differentiation, corresponding to the up-regulation of p21(WAF1/cip1). Moreover, butyrate-induced p21 expression was linked to phosphorylation of JNK1. Reduced JNK1 expression by small interfering RNA suppressed butyrate-induced apoptosis. We concluded that JNK1 plays a critical role in the regulation of homeostasis and in the suppression of tumor formation in the intestine, which was linked to the altered expression of p21(WAF1/cip1).     

Aneuploidy arises at early stages of Apc-driven intestinal tumorigenesis and pinpoints conserved chromosomal loci of allelic imbalance between mouse and human

Although chromosomal instability characterizes the majority of human colorectal cancers, the contribution of genes such as adenomatous polyposis coli (APC), KRAS, and p53 to this form of genetic instability is still under debate. Here, we have assessed chromosomal imbalances in tumors from mouse models of intestinal cancer, namely Apc(+/1638N), Apc(+/1638N)/KRAS(V12G), and Apc(+/1638N)/Tp53-/-, by array comparative genomic hybridization. All intestinal adenomas from Apc(+/1638N) mice displayed chromosomal alterations, thus confirming the presence of a chromosomal instability defect at early stages of the adenoma-carcinoma sequence. Moreover, loss of the Tp53 tumor suppressor gene, but not KRAS oncogenic activation, results in an increase of gains and losses of whole chromosomes in the Apc-mutant genetic background. Comparative analysis of the overall genomic alterations found in mouse intestinal tumors allowed us to identify a subset of loci syntenic with human chromosomal regions (eg, 1p34-p36, 12q24, 9q34, and 22q) frequently gained or lost in familial adenomas and sporadic colorectal cancers. The latter indicate that, during intestinal tumor development, the genetic mechanisms and the underlying functional defects are conserved across species. Hence, our array comparative genomic hybridization analysis of Apc-mutant intestinal tumors allows the definition of minimal aneuploidy regions conserved between mouse and human and likely to encompass rate-limiting genes for intestinal tumor initiation and progression.     

Helicobacter hepaticus infection promotes colon tumorigenesis in the BALB/c-Rag2(-/-) Apc(Min/+) mouse

Adenomatous polyposis coli (APC) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6-Apc(Min/+)) congenic strain was generated by backcrossing into BALB/c the Apc(Min) allele from C57BL/6J-Apc(Min/+) mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg-Rag2(-/-) Apc(Min/+)) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus-infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus-infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus, which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.     

Loss of nuclear p21(Cip1/WAF1) during neoplastic progression to metastasis in gamma-irradiated p21 hemizygous mice

p21(Cip1/WAF1) localizes to the nucleus in response to gamma-irradiation induced DNA damage and mediates a G(1) checkpoint arrest. Although gamma-irradiated p21(+/-) mice develop a broad spectrum of tumors, gamma-irradiated p21(-/-) mice develop significantly more metastatic cancers. To evaluate the expression of p21 in tissues prone or resistant to tumorigenesis as a function of gamma-irradiation, and to determine whether phenotypic loss of p21 heterozygosity occurs during tumor progression in p21(+/-) mice, tissues and tumors from gamma-irradiated mice were evaluated immunohistochemically. The percentage of tumors in p21(+/-) mice that were nuclear p21-positive declined with progression to metastasis (p<0.0001). Benign tumors were more often p21-positive and comprised of larger subsets of nuclear p21-positive cells than were malignant tumors of the same histopathological type, while metastatic cancers were nuclear p21-negative (p=0.0003). Even when a primary cancer was comprised of a subset of nuclear p21-positive cells, the metastatic foci of that same cancer were nuclear p21-negative. Mesenchymal tumors, though rare, were more likely metastatic than were epithelial tumors (p=0.0004), and these were invariably nuclear p21-negative. Prepubescent epithelial tissues from which most tumors later originated in mice with reduced p21 gene dosage (i.e., harderian gland, ovary, small intestine, and lung) were p21 expressive within 4 h of gamma-irradiation (p=0.0625), so that p21/Ki67 ratios increased post-gamma-irradiation (p=0.03). In contrast, p21 did not localize to nuclei of cortical thymocytes, a tissue where tumorigenesis was not augmented by reduced p21 gene dosage. Cellular subclones of malignant tumors, especially those of mesenchymal cell origin, which lack nuclear p21 may more readily acquire the genetic alterations of the metastatic phenotype.     

Somatic Apc mutations are selected upon their capacity to inactivate the beta-catenin downregulating activity

The APC gene, originally identified as the gene for familial adenomatous polyposis (FAP), is now considered as the true "gatekeeper" of colonic epithelial proliferation. Its main tumor suppressing activity seems to reside in the capacity to properly regulate intracellular beta-catenin signaling. Most somatic APC mutations are detected between codons 1286 and 1513, the mutation cluster region (MCR). This clustering can be explained either by the presence of mutation-prone sequences within the MCR, or by the selective advantage provided by the resulting truncated polypeptides. Here, a Msh2-deficient mouse model (Msh2(delta 7N) ) was generated and bred with Apc(1638N) and Apc(Min) that allowed the comparison of the somatic mutation spectra along the Apc gene in the different allelic combinations. Mutations identified in Msh2(delta 7N/delta 7N) tumors are predominantly dinucleotide deletions at simple sequence repeats leading to truncated Apc polypeptides that partially retain the 20 a.a. beta-catenin downregulating motifs. In contrast, the somatic mutations identified in the wild type Apc allele of Msh2(delta 7N/delta 7N) /Apc(+/1638N) and Msh2(delta 7N/delta 7N) /Apc(+/Min) tumors are clustered more to the 5' end, thereby completely inactivating the beta-catenin downregulating activity of APC. These results indicate that somatic Apc mutations are selected during intestinal tumorigenesis and that inactivation of the beta-catenin downregulating function of APC is likely to represent the main selective factor.     

Cisplatin-DNA damage in p21WAF1/Cip1 deficient mouse keratinocytes exposed to cisplatin

In response to DNA damage, cell cycle arrest, apoptosis, and DNA repair are mediated by a TP53 pathway that induces p21(WAF1/Cip1). The chemotherapeutic drug cis-diamminedichloroplatinum-II (cisplatin) damages cellular DNA by forming cis-diammineplatinum-N(7)-d[GpG] and cis-diammine-platinum-N(7)-d[ApG] adducts. To investigate the role of p21, skin keratinocytes from p21(WAF1/Cip1) wild-type (+/+), heterozygous (+/-), and null (-/-) mice, cultured in calcium levels designed to maintain a proliferating state, were exposed to 5 microM cisplatin continuously for 0, 8, 24, 48 and 72 h. At all time points the (+/-) cells had the fewest Pt-DNA adducts, and at 24 h mean Pt-DNA adduct levels were 541, 153 and 779 fmol adduct/mug DNA for p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively [P < 0.05 for (+/+) versus (+/-) and (-/-) versus (+/-)]. In order to understand underlying events, we examined p21(WAF1/Cip1) messenger RNA (mRNA), cell cycle arrest, and apoptosis in these cells. At 48 h of cisplatin exposure p21(WAF1/Cip1) mRNA expression was 2-fold higher in the (+/+) cells, compared to the (+/-) cells. At 24 h, the % of cells in S-phase in cisplatin-exposed cultures, compared to unexposed cultures, was decreased by 51, 40 and 11% in p21(WAF1/Cip1) (+/+), (+/-) and (-/-) cells, respectively (P = 0.04, ANOVA). At 24, 48 and 72 h the % of cisplatin-exposed (+/+) cells in apoptosis was 9.4-10.5%, while the cisplatin-exposed (+/-) and (-/-) cells had 1.2-3.7% of cells in apoptosis. The data support the interpretation that DNA replication arrest and apoptosis do not completely explain the low levels of Pt-DNA adducts in the (+/-) cells, and suggest that p21(WAF1/Cip1) controls activity resulting in either low Pt-DNA adduct formation or enhanced Pt-DNA adduct removal.     

The genetic background modifies the spontaneous and X-ray-induced tumor spectrum in the Apc1638N mouse model.

The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.     

Overexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline ovarian tumors

Ovarian epithelial tumors are classically divided into benign, malignant, and borderline or of low malignant potential. It is controversial whether this last group of tumors should be considered benign or malignant. Expression of cell cycle markers has recently been linked to tumor behavior and response to treatment. It has been shown that one of the pathways through which the p53 gene controls the cell cycle is by transactivating p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor. By inhibiting cdks, p21WAF1/CIP1 blocks the G-1 to S-phase transition in the cell cycle. p53 can be regulated by MDM2 (murine double minute-2) through direct inactivation or promotion of its cytoplasmic degradation. In an attempt to investigate the cell cycle checkpoint mechanisms of these tumors, we studied the expression of p53, Ki-67, MDM2, and p21WAF1/CIP1 by immunohistochemistry. We analyzed the expression of these proteins in 19 cystadenomas (8 serous and 11 mucinous), 40 borderline tumors (31 serous and 9 mucinous), and 18 serous carcinomas of the ovary. p21WAF1/CIP1 was expressed in 7 of 19 (37%) benign cystadenomas, 32 of 40 (80%) borderline tumors (93.5% of serous and 33% of mucinous), and in 9 of 18 (50%) serous carcinomas. Ki-67 was only weakly expressed in 8 of 19 (42%) benign cystadenomas, all borderline tumors showed Ki-67 staining in less than 50% of the cells, and 55% of serous carcinomas stained in more than 50% of tumor cells. p53 was absent in all but 1 of the cystadenomas, was expressed in 9 of 40 (22.5%) borderline tumors (25.8% of serous and 11% of mucinous), and in 10 of 18 (55%) carcinomas. All 11 implants of serous borderline tumors expressed p21WAF1/CIP1. Most serous borderline tumors expressed higher levels of MDM2 compared with the benign cystadenomas and carcinomas. Four of the serous borderline implants (40%) expressed MDM2. Coexpression of p21WAF1/CIP1 and MDM2 characterizes serous borderline tumors of the ovary and their implants, which suggests that these cell cycle control proteins are important in these tumors and may be related to tumor progression. Low expression of p53 protein in serous borderline tumors might be in part mediated by MDM2. This suggests that the p53 pathway is intact in most of these tumors, in contrast with carcinomas, in which high expression of p53 has been related to mutations of this gene.     

Colorectal cancers in a new mouse model of familial adenomatous polyposis: influence of genetic and environmental modifiers

Murine models of familial adenomatous polyposis harbor a germinal heterozygous mutation on Apc tumor suppressor gene. They are valuable tools for studying intestinal carcinogenesis, as most human sporadic cancers contain inactivating mutations of APC. However, Apc(+/-) mice, such as the well-characterized Apc(Min/+) model, develop cancers principally in the small intestine, while humans develop mainly colorectal cancers. We used a Cre-loxP strategy to achieve a new model of germline Apc invalidation in which exon 14 is deleted. We compared the phenotype of these Apc(Delta14/+) mice to that of the classical Apc(Min/+). The main phenotypic difference is the shift of the tumors in the distal colon and rectum, often associated with a rectal prolapse. Thus, the severity of the colorectal phenotype is partly due to the particular mutation Delta14, but also to environmental parameters, as mice raised in conventional conditions developed more colon cancers than those raised in pathogen-free conditions. All lesions, including early lesions, revealed Apc LOH and loss of Apc gene expression. They accumulated beta-catenin, overexpressed the beta-catenin target genes cyclin D1 and c-Myc, and the distribution pattern of glutamine synthetase, a beta-catenin target gene recently identified in the liver, was mosaic in intestinal adenomas. The Apc(Delta14/+) model is thus a useful new tool for studies on the molecular mechanisms of colorectal tumorigenesis.     

The C-terminal domain of the adenomatous polyposis coli (Apc) protein is involved in thyroid morphogenesis and function

Adenomatous polyposis coli (APC) is a multifunctional protein as well as a tumor suppressor. To determine the functions of the C-terminal domain of Apc, we have investigated Apc ( 1638T/1638T ) mice, which express a truncated Apc that lacks the C-terminal domain. Apc ( 1638T/1638T ) mice are tumor free and exhibit growth retardation. In the present study, we analyzed the morphology and functions of the thyroid gland in Apc ( 1638T/1638T ) mice. There was no significant difference in the basal concentration of serum thyroid hormones between Apc ( 1638T/1638T ) and Apc (+/+) mice. Thyroid follicle size was significantly larger in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. The extent of serum T4 elevation following exogenous thyroid-stimulating hormone (TSH) injection was lower in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. TSH also induced a greater reduction in thyroid follicle size in Apc ( 1638T/1638T ) mice than in Apc (+/+) mice. Analyses using immunohistochemistry and electron microscopy indicated that follicular epithelial cells in Apc ( 1638T/1638T ) mice had an enlarged rough endoplasmic reticulum of irregular shape. These results suggest that the C-terminal domain of Apc is involved in thyroid morphology and function.     

Efficacy of chemopreventive agents for growth inhibition of Apc [+/-] 1638NCOL colonic epithelial cells

Germline mutations of the Apc tumor suppressor gene result in increased risk for gastrointestinal carcinogenesis. The Apc1638N [+/-] mouse exhibits accelerated gastrointestinal carcinogenesis that is modifiable by select pharmacological and dietary agents. Experiments in the present study were conducted on a subculturable epithelial 1638NCOL cell line established from histologically normal colon of Apc1638N [+/-] mouse to examine the effects of selected chemopreventive agents that differ in their mechanism of action. Extent of growth arrest, number of cell population doublings, cell cycle progression and aneuploid G0/G1: S + G2/M ratio represented the quantitative endpoints for the susceptibility and efficacy of chemopreventive agents. Treatment of exponentially growing 1638NCOL cells with maximum cytostatic dose of 9cisRA, DFMO or SUL (100 microM) produced a 60-70% growth arrest, that with TAM and AMF (10 microM) produced a 20-40% growth arrest, while that with OLT (100 microM) produced a 25% growth arrest. This response was associated with corresponding decrease in the number of cell population doubling. 9cisRA, SUL or AMF increased the aneuploid G0/G1: S + G2/M ratio by inducing G1 checkpoint arrest, while DFMO, TAM and OLT decreased the ratio by inducing G2 checkpoint arrest. Thus, cell cycle phase-dependent susceptibility of the Apc [+/-] 1638NCOL cell line to mechanistically distinct chemopreventive agents validates a novel colon epithelial cell culture model for mechanistic, preventive or therapeutic studies on Apc regulated colon carcinogenesis.     

Patterns of p21waf1/cip1 expression in non-papillomatous nasal mucosa, endophytic sinonasal papillomas, and associated carcinomas

AIMS: To clarify p21(waf1/cip1) expression in sinonasal lesions. METHODS: Archived surgical specimens from 38 patients were investigated by means of immunohistochemistry. p21(waf1/cip1) staining was evaluated in the different layers of the epithelium. In addition, human papillomavirus (HPV) infection and p53 protein overexpression were assessed and correlated with p21(waf1/cip1) expression. RESULTS: p21(waf1/cip1) staining was negative in non-papillomatous nasal mucosa. HPV infection and p53 protein overexpression were not seen. Sixteen of 20 inverted papillomas showed p21(waf1/cip1) expression. HPV infection was found in 16 cases and p53 protein overexpression was present in 13 specimens. Expression of p21(waf1/cip1) was restricted to surface cells in five cases, but involved basal/parabasal cells in 11 specimens. Immunoreactivity for p21(waf1/cip1) in basal/parabasal cells colocalised with p53 protein overexpression. Enhanced expression rates for p21(waf1/cip1) were seen in transitional and squamous epithelium compared with columnar epithelium. p21(waf1/cip1) expression involved only surface cells in cylindrical cell papillomas. HPV infection and p53 protein overexpression were detected in all specimens. One of five squamous cell carcinomas showed p21(waf1/cip1) expression. HPV infection was seen in two cases, and all carcinomas showed p53 protein overexpression. CONCLUSIONS: Expression of p21(waf1/cip1) is associated with terminal differentiation in surface cells in inverted papillomas and cylindrical cell papillomas, but not in non-papillomatous nasal mucosa. Overexpression of p53 protein colocalises with p21(waf1/cip1) expression in basal/parabasal cells in inverted papillomas but not in cylindrical cell papillomas. Expression of p21(waf1/cip1) in squamous cell carcinomas involves a subset of tumours with p53 protein overexpression.     

p53 expression in CMV-infected cells: association with the alternative expression of the p53 transactivated genes p21/WAF1 and MDM2

The p53 tumour suppressor gene is a cell cycle regulator, able to induce cell cycle arrest to allow DNA repair or apoptosis. The molecular mechanisms underlying p53 action imply transactivation of p53 dependent genes such as WAF1 (for wild type p53 associated fragment 1) and the murine double minute (MDM2) gene. In some cases, inactivation of the p53 gene results from p53 gene mutations leading to p53 protein accumulation, but in others it may results from mechanisms other than mutation, such as interaction with viral or cellular proteins. The expression of p53 protein and p53 transactivated gene proteins p21/WAF1 and MDM2, combined with in situ detection of apoptosis, was studied in specimens of CMV-infected patients as an in vivo model of p53 alteration not due to point mutation. p53 positivity was found in CMV + cells in different tissues, in cells with typical inclusion bodies, and in in situ hybridization and immunohistochemistry CMV + cells without inclusions (hidden infection). Although this p53 reactivity was accompanied by the expression of MDM2 and p21/WAF1 proteins, the patterns of MDM2 and p21/WAF1 protein expression were mutually exclusive, and were associated with the presence or absence of inclusion bodies. Nuclei bearing inclusion bodies were usually MDM2 +, p21/WAF1?, while hidden infected cells were usually MDM2?, p21/WAF1 +. Apoptosis was not detected in any tissue section from CMV-infected patients. Two alternative patterns were found in CMV-infected tissues: p53 +, p21/WAF1 +, MDM2?, or p53 +, p21/WAF1?, MDM2 + protein expression. These may represent examples of p53 dependent alternative effects in the course of CMV infection. Early stages are represented by CMV + cells without inclusion bodies, which display p53 and p21/WAF1 expression, suggesting that p53 could be acting as a growth suppressor protein. Late CMV infection is represented by cells harbouring inclusion bodies. These cells showed a p53 +, p21/WAF1?, MDM2 + profile, consistent with MDM2 mediated p53 inactivation. The absence of p21/WAF1 expression and lack of apoptosis suggest that the p53 protein expressed by MDM2 + cells could be functionally inactivated in CMV-infected cells with inclusion bodies. Previous studies have suggested that p53 inactivation by MDM2 over-expression occurs in sarcomas and lymphomas. Our observations seem to indicate that this mechanism of MDM2 mediated p53 inactivation may play a role in the late phase of CMV infection.     

Limited effects of Muc1 deficiency on mouse adenovirus type 1 respiratory infection

Muc1 (MUC1 in humans) is a membrane-tethered mucin that exerts anti-inflammatory effects in the lung during bacterial infection. Muc1 and other mucins are also likely to form a protective barrier in the lung. We used mouse adenovirus type 1 (MAV-1, also known as MAdV-1) to determine the role of Muc1 in the pathogenesis of an adenovirus in its natural host. Following intranasal inoculation of wild type mice, we detected increased TNF-α, a cytokine linked to Muc1 production, but no consistent changes in the production of lung Muc1, Muc5ac or overall lung mucus production. Viral loads were modestly higher in the lungs of Muc1(-/-) mice compared to Muc1(+/+) mice at several early time points but decreased to similar levels by 14 days post infection in both groups. However, cellular inflammation and the expression of CXCL1, CCL5, and CCL2 did not significantly differ between Muc1(-/-) and Muc1(+/+) mice. Our data therefore suggest that Muc1 may contribute to a physical barrier that protects against MAV-1 respiratory infection. However, our data do not reveal an anti-inflammatory effect of Muc1 that contributes to MAV-1 pathogenesis.     

Glycoconjugate metabolism in a cystic fibrosis knockout mouse model

Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.     

Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.     

Expression of p21 WAF1/CIP1 protein in colorectal cancers: study of its relation to p53 mutation and Ki67 antigen expression

Mutations of the p53 gene are the most common genetic alteration in malignant human tumors. A cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is thought to be an important mediator of p53-induced cell cycle arrest. Although numerous studies have reported p53 expression and mutation in colorectal cancer few of them have correlated p53 expression with that of its downstream effector p21 and with the proliferation index as measured by expression of the Ki67 nuclear antigen. We studied p53, p21 and Ki67 expression by immunohistochemistry and molecular biology in 35 colorectal carcinomas. We compared these findings with each other and with clinical factors. Sixty three percent of tumors expressed p53 whereas seventy one percent expressed p21WAF1/CIP1. In adenocarcinomas, p21 staining was heterogeneous: p21-reactive cells were seen in the most differentiated areas. There was no correlation between p21WAF1/CIP1 and p53 expression, p53 mutation, Ki67 expression or clinical factors such as sex or location of the tumor. On the other hand, there was a statistical relationship between p21 expression and survival: our results indicated an association between high p21 expression and lower stages p21WAF1/CIP1 appears to be induced independently of p53 in these tumors and may be associated with differentiation rather than proliferation.     

Spontaneous aberrant crypt foci in Apc1638N mice with a mutant Apc allele

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.