p53 expression and DNA ploidy pattern in Egyptian colorectal carcinoma

BACKGROUND/AIMS: p53 gene mutation occurs in about 50-60% of colorectal carcinoma cases. This mostly occurs as a late event in the adenoma-carcinoma sequence. These late stages are associated with more aneuploidy compared to adenomas and early carcinomas. However there is a controversy regarding the relation between p53 overexpression and DNA index. This study was designed to investigate the relationship between p53 status and DNA ploidy pattern. METHODOLOGY: Nuclear DNA content of paraffin-embedded material from 83 colectomy specimens for colorectal carcinoma was measured by flow cytometry. Also, p53 was detected by immunohistochemistry in 73 out of the 83 tumor cases using a monoclonal antibody that detects both wild and mutant p53 proteins (Biogenex 1801). RESULTS: Aneuploidy was identified in 37 cases (46.25%). Tumors with rectal location were significantly more aneuploid in comparison to other sites (P = 0.009), p53 staining showed three patterns: diffuse staining (29 cases), focal (13 cases), and negative (31 cases). Diffuse p53 staining was associated with aneuploidy (P = 0.04). The majority of DNA indices fell within the range 1.1-2.2 (32 out of 37). Twenty-one of these had DNA index = 1.1-1.8 (aneuploidy short of tetraploidy) significantly associated with diffuse p53 staining compared with peritetraploid cases (DNA index 1.8-2.2) (P = 0.034). CONCLUSIONS: p53 immunohistochemistry demonstrates two distinct patterns in colorectal carcinoma. Diffuse p53 staining, which is associated with aneuploidy short of tetraploidy (DNA index 1.1-1.8), a finding which is different from previously published work. Focal p53 staining pattern, in contrast, is related to high G2M and more abnormal tetraploid peaks but less aneuploidy.     

Human papillomavirus 16 E6 expression disrupts the p53-mediated cellular response to DNA damage.

Infection with certain types of human papillomaviruses (HPV) is highly associated with carcinomas of the human uterine cervix. However, HPV infection alone does not appear to be sufficient for the process of malignant transformation, suggesting the requirement of additional cellular events. After DNA damage, normal mammalian cells exhibit G1 cell-cycle arrest and inhibition of replicative DNA synthesis. This mechanism, which requires wild-type p53, presumably allows cells to undertake DNA repair and avoid the fixation of mutations. We directly tested whether the normal response of cervical epithelial cells to DNA damage may be undermined by interactions between the E6 protein expressed by oncogenic HPV types and wild-type p53. We treated primary keratinocytes with the DNA-damaging agent actinomycin D and demonstrated inhibition of replicative DNA synthesis and a significant increase in p53 protein levels. In contrast, inhibition of DNA synthesis and increases in p53 protein did not occur after actinomycin D treatment of keratinocytes immortalized with HPV16 E6/E7 or in cervical carcinoma cell lines containing HPV16, HPV18, or mutant p53 alone. To test the effects of E6 alone on the cellular response to DNA damage, HPV16 E6 was expressed in the carcinoma cell line RKO, resulting in undetectable baseline levels of p53 protein and loss of the G1 arrest that normally occurs in these cells after DNA damage. These findings demonstrate that oncogenic E6 can disrupt an important cellular response to DNA damage mediated by p53 and may contribute to the subsequent accumulation of genetic changes associated with cervical tumorigenesis.     

Human papillomavirus DNA and p53 status in stage IB bulky cervical cancer

Summary We used the polymerase chain reaction (PCR) to study the presence and typing of human papillomavirus (HPV) DNA, and PCR/single-strand confirmation polymorphism to survey the mutations of the p53 gene in exons 5–9 in 26 bulky stage IB cervical cancers. The HPV DNA was present in 20 out of the 21 (95%) squamous-cell carcinoma tissues, including 13 cases of HPV-16, 0 case of HPV-18, and 7 cases of other HPV types. In the other 5 adenocarcinoma tissues, 3 had HPV type-18 DNA and 2 had no detectable HPV DNA. The distribution of HPV DNA in bulky tumors was not statistically different from that in the other 228 squamous-cell carcinomas and 23 adencarcinomas of smaller size operated upon during the same period. Out of the 26 patients, 9 (35%) had lymph node metastasis at the time of operation. During the follow-up period ranging from 19 to 48 months, 9 patients had recurrence and 7 of them died of disease. The distribution of HPV types was not related to the prognosis in these patients. There were no p53 mutations detected in all the 26 samples. Thus, HPV type and the status of p53 could not serve as additional prognostic factors in stage IB bulky cervical cancers.Abbreviations HPV human papillomavirus - PCR-SSCP polymerase chain reaction/single-strand confirmation polymorphism Funded by a grant from the National Taiwan University Hospital NTUH-82070-B26     

Quaternary structures of tumor suppressor p53 and a specific p53 DNA complex

The homotetrameric tumor suppressor p53 consists of folded core and tetramerization domains, linked and flanked by intrinsically disordered segments that impede structure analysis by x-ray crystallography and NMR. Here, we solved the quaternary structure of human p53 in solution by a combination of small-angle x-ray scattering, which defined its shape, and NMR, which identified the core domain interfaces and showed that the folded domains had the same structure in the intact protein as in fragments. We combined the solution data with electron microscopy on immobilized samples that provided medium resolution 3D maps. Ab initio and rigid body modeling of scattering data revealed an elongated cross-shaped structure with a pair of loosely coupled core domain dimers at the ends, which are accessible for binding to DNA and partner proteins. The core domains in that open conformation closed around a specific DNA response element to form a compact complex whose structure was independently determined by electron microscopy. The structure of the DNA complex is consistent with that of the complex of four separate core domains and response element fragments solved by x-ray crystallography and contacts identified by NMR. Electron microscopy on the conformationally mobile, unbound p53 selected a minor compact conformation, which resembled the closed conformation, from the ensemble of predominantly open conformations. A multipronged structural approach could be generally useful for the structural characterization of the rapidly growing number of multidomain proteins with intrinsically disordered regions.     

c-myc and bcl-2 modulate p53 function by altering p53 subcellular trafficking during the cell cycle.

We have studied the ability of c-myc and bcl-2 oncogenes to modulate p53 function. Our studies show that coincident expression of human Bcl-2 protein with p53 prolongs survival of murine erythroleukemia cells. This effect was associated with a loss of the G1 specificity of p53-mediated cell cycle arrest. Furthermore, we found that the c-myc and bcl-2 genes cooperate to inhibit p53 functions. Coexpression of bcl-2 and c-myc can totally overcome p53-induced apoptosis and cell cycle arrest by altering the subcellular trafficking of p53 during the cell cycle: the p53 remains in the cytoplasm of the cotransfected cells during a critical period in G1. This finding suggests a mechanism by which normal hematopoietic progenitors can survive and proliferate despite p53 expression and by which the inappropriate expression of bcl-2 and c-myc can cooperate in transformation.     

p53 gene mutations in soft-tissue sarcomas-correlations with p53 immunohistochemistry and DNA ploidy

The significance ofp53 mutations in a group of 67 soft-tissue tumors was examined using single-strand conformation polymorphism and direct sequencing analysis. Molecular findings were correlated with immunohistochemical detection of thep53 protein and DNA ploidy status. Mutations of thep53 gene were detected in 13 (19.5%) out of 67 cases of soft-tissue tumors. Only there were localized outside the conservative regions of thep53 gene. Six mutations were described for the first time in these tumors. Most of the mutations were point mutations in exons 5–8 and, in one case, a deletion at the 3-splice site of exon 5 could be demonstrated. There was no significant correlation between the occurrence ofp53 mutations and the histological grade, although a high number of mutations were defined in poorly differentiated tumors (grade 3). Molecular finding of ap53 gene mutation and immunohistochemical detection ofp53 expression did not correlate, which may be due to the high percentage of nonsense mutations in our study (50%). We confirm that only DNA sequencing allows a unique identification and differentiation of mutations in thep53 gene. Other factors may be responsible for the detection ofp53 protein in many cases. Histological grade correlated with aneuploidy. The frequency of mutations observed was in accordance with values quoted in the literature. Generally,p53 mutations andp53 overexpression are more likely to represent a late event in the oncogenesis of soft-tissue tumors.     

Anti-p53 antibodies and p53 protein expression in cholangiocarcinoma

BACKGROUND/AIMS: Mutations of p53 are found in the majority of human malignancies. The accumulated mutant p53 can be detected in tumor sections by immunohistochemical methods. The abnormal accumulation of the defective p53 protein can induce the host to develop anti-p53 antibodies in sera of cancer patients. This study aimed to investigate the presence of anti-p53 antibodies in sera of patients with cholangiocarcinoma and to evaluate the correlation between such antibodies and p53 protein accumulation. METHODOLOGY: The presence of serum anti-p53 antibodies in 49 patients with cholangiocarcinoma was determined by ELISA kit (Pharma Cell, France). Immunohistochemical detection of p53 protein expression was examined in available tissue samples of 36 patients. RESULTS: Serum anti-p53 antibodies were detected in 6 of 49 patients with cholangiocarcinoma (12.2%). Immunostaining of p53 was found in 15 of 36 patients (41.6%) and 4 of these 15 patients (26.7%) were positive for anti-p53 antibodies. The association between anti-p53 antibodies and p53 protein expression was statistically significant (P=0.023). No correlation was found between the presence of anti-p53 antibodies and sex, age, histological grade, site and stage of tumor (P>0.05). CONCLUSIONS: The majority of serum anti-p53 antibodies detected in cholangiocarcinoma were specifically associated with the accumulation of p53 protein in tumor tissues. However, antibody generation against the p53 protein is a relatively uncommon event in cholangiocarcinoma.     

In vivo stabilization of the Dnmt1 (cytosine-5)- methyltransferase protein

The Dnmt1o form of the Dnmt1 (cytosine-5)-methyltransferase enzyme is synthesized and stored in the cytoplasm of the oocyte and is used after fertilization to maintain methylation patterns on imprinted genes. After implantation of the blastocyst, Dnmt1o is replaced by the Dnmt1 form, which has an additional 118 aa at its amino terminus. To investigate functional differences between Dnmt1o and Dnmt1, mice were generated with a mutant allele, Dnmt1(V), which synthesized Dnmt1o instead of Dnmt1 in all somatic cells. Homozygous Dnmt1(V) mice were phenotypically normal, and had normal levels of genomic methylation, indicating that Dnmt1o adopts the maintenance methyltransferase function of Dnmt1. Despite the apparent equivalence of Dnmt1o and Dnmt1 maintenance methyltransferase function in somatic cells, the Dnmt1o protein was found at high levels (with a corresponding high enzymatic activity) in Dnmt1(V) mice. In heterozygous Dnmt1(V)/+ embryonic stem cells and early embryos, equal steady-state levels of Dnmt1o and Dnmt1 proteins were produced from the Dnmt1(V) and the WT Dnmt1 alleles, respectively. However, in older embryos and adults, the Dnmt1(V) allele produced five times the steady-state level of protein of the WT Dnmt1 allele. The difference in Dnmt1o and Dnmt1 levels is due to a developmentally regulated mechanism that degrades the Dnmt1 protein. The intrinsic stability of the Dnmt1o protein is the most likely reason for its use as a maternal-effect protein; stable ooplasmic stores of Dnmt1o would be available to traffick into the nuclei of the eight-cell stage embryo and maintain methylation patterns on alleles of imprinted genes during the fourth embryonic S phase.     

Inhibition of p53-induced apoptosis without affecting expression of p53-regulated genes

Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene expression. Expression of various p53 targets implicated as mediators of p53-induced apoptosis was also not affected by IL-6 or TG. These compounds thus can bypass the effect of wild-type p53 on gene expression and inhibit apoptosis. IL-6 and TG activated different p53-independent pathways of gene expression that include up-regulation of antiapoptotic genes. IL-6 and TG also activated different differentiation-associated genes. The ability of compounds such as cytokines and calcium mobilizers to inhibit p53-mediated apoptosis without generally inhibiting gene expression regulated by p53 can facilitate tumor development and tumor resistance to radiation and chemotherapy in cells that retain wild-type p53.     

p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting

DNA binding activity of p53 is crucial for its tumor suppressor function. Our recent studies have shown that four molecules of the DNA binding domain of human p53 (p53DBD) bind the response elements with high cooperativity and bend the DNA. By using A-tract phasing experiments, we find significant differences between the bending and twisting of DNA by p53DBD and by full-length human wild-type (wt) p53. Our data show that four subunits of p53DBD bend the DNA by 32-36 degrees, whereas wt p53 bends it by 51-57 degrees. The directionality of bending is consistent with major groove bends at the two pentamer junctions in the consensus DNA response element. More sophisticated phasing analyses also demonstrate that p53DBD and wt p53 overtwist the DNA response element by approximately 35 degrees and approximately 70 degrees, respectively. These results are in accord with molecular modeling studies of the tetrameric complex. Within the constraints imposed by the protein subunits, the DNA can assume a range of conformations resulting from correlated changes in bend and twist angles such that the p53-DNA tetrameric complex is stabilized by DNA overtwisting and bending toward the major groove at the CATG tetramers. This bending is consistent with the inherent sequence-dependent anisotropy of the duplex. Overall, the four p53 moieties are placed laterally in a staggered array on the external side of the DNA loop and have numerous interprotein interactions that increase the stability and cooperativity of binding. The novel architecture of the p53 tetrameric complex has important functional implications including possible p53 interactions with chromatin.     

p53 gene mutations and expression of p53 and mdm2 proteins in invasive breast carcinoma

The aim of the study was to analyzep53 gene mutations and the expression of p53 and mdm2 proteins in 31 randomly selected invasive breast carcinomas. The results were then correlated with tumor grade, stage, estrogen receptor status, nodal status, and DNA ploidy. The expression of the proteins p53 and mdm2 was determined immunohistochemically using formalin-fixed, paraffin-embedded material. Screening for p53 mutation involved analysis of the highly conserved regions of thep53 gene (exons 5–9) by the polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) technique. PCR products with band shifts were directly sequenced. Immunohistochemical staining of p53 was positive in 9 cases (29.0%), only 2 of which showed ap53 gene mutation. These were identified as a CG transversion at the second position of codon 278 in exon 8 and an AG transition at the second position of codon 205 in exon 6. A third case with a mutation was observed (CT transition, position 1 of codon 250 in exon 7) that did not show p53 immunohistochemically. Of the 9 p53-positive tumors, 2 were moderately differentiated (grade II). The remaining tumors were poorly differentiated (7/9). By contrast, p53-negative carcinomas were well differentiated (grade I) in most cases (P=0.02). DNA cytometry in 8 of the 9 p53-positive carcinomas revealed an aneuploid stem line. The majority of the p53-negative tumors were diploid (P=0.01). Mdm2 oncoprotein was detected in 10 tumors (32.2%), 4 of which were p53-positive, including the 3 with mutations. The grading of the mdm2-positive tumors was moderate or poor, G1 carcinomas were always noted to be mdm2-negative (P=0.04). Overexpression of p53 protein is a complex mechanism and does not merely indicate the detection of mutations in thep53 gene. This study has shown that p53 expression correlates with tumor grade and DNA ploidy. Mdm2 expression was also associated with the tumor grade. Immunohistological demonstration of the p53 protein alone is insufficient as a basis for comment on the functional state of thep53 gene and gene product. The interrelation between recognition of the p53 protein and gene mutation needs more careful assessment to define their roles in the control of neoplasia.     

Folic acid and its metabolites modulate IGF-I receptor gene expression in colon cancer cells in a p53-dependent manner

The insulin-like growth factor-I receptor (IGF-IR) has an important role in colorectal cancer development and progression. IGF-IR displays a potent anti-apoptotic activity and is overexpressed in primary tumors and colon cancer-derived cell lines. Folic acid, a member of the vitamin B family, is a chemopreventive agent whose deficiency has been linked to an enhanced colon cancer risk. The present study was aimed at testing the hypothesis that part of the modulatory effect of folic acid on malignant transformation may be attributed to its ability to regulate IGF-IR gene expression. Regulation of IGF-IR gene expression by folic acid was assessed using western blots, RT-PCR, transient transfections and chromatin immunoprecipitation assays. Activation of the IGF-IR signaling pathway was evaluated by measuring phosphorylation of ERK, and apoptosis was assayed using poly (ADP-ribose) polymerase cleavage and annexin V-FITC staining. Results obtained showed that folic acid induced a dose-dependent decrease in IGF-IR protein and mRNA levels in the HCT116 +/+ colon cancer cell line. This effect was associated with a significant reduction in IGF-IR promoter activity. Similar effects were elicited by the folic acid metabolites dihydrofolic acid and tetrahydrofolic acid. In addition, folic acid abrogated the IGF-I-stimulated phosphorylation of the downstream signaling molecule ERK1/2 and exhibited a pro-apoptotic activity. Moreover, folic acid induced a significant decrease in Sp1 binding to the IGF-IR promoter region. Finally, folic acid had no effect in wild-type p53-depleted HCT116 -/- and Caco-2 cells. In conclusion, the mechanism of action of folic acid involves regulation of IGF-IR gene expression. The ability of folic acid to downregulate the IGF-I signal transduction pathway may allow the micronutrient to function as a chemopreventive agent. Folic acid deficiency, on the other hand, may lead to increased IGF-IR gene expression, with ensuing pathological activation by endocrine and/or autocrine/paracrine IGF-I.     

Neuropeptide-Y Y5 receptors modulate the onset and maintenance of operant ethanol self-administration

BACKGROUND: Neuropeptide Y (NPY) is the most abundant and widely distributed peptide in the mammalian central nervous system and increases feeding behavior at NPY Y1 or Y5 receptor subtypes. Recent pharmacological and mutant mouse data indicate that NPY activity at its receptors can influence ethanol self-administration, although the direction and strength of this influence are not clear. METHODS: Effects of the novel NPY Y5 receptor antagonist L-152,804 on the onset and maintenance of operant self-administration were examined in male C57BL/6J mice, which were trained to self-administer ethanol (10% v/v) versus water via the sucrose substitution method during 16 hr overnight sessions. After 4 months of baseline responding, mice were injected with L-152,804 (0, 10, 30, or 60 mg/kg, intraperitoneally) before operant sessions. Potential locomotor effects of L-152,804 and possible interaction with the sedative properties of ethanol also were examined. RESULTS: All three doses of L-152,804 significantly delayed the onset of ethanol-reinforced responding relative to vehicle injection. L-152,804 produced no effect on the total number of ethanol- or water-reinforced responses per 16 hr session. However, L-152,804 selectively modulated the temporal distribution of ethanol-reinforced responding depending on the dose (10 and 60 mg/kg) and time point measured in a manner consistent with blockade of ethanol reinforcement. Additional experiments determined that L-152,804 (10 or 60 mg/kg) did not alter spontaneous locomotor activity or influence the sedative effects of ethanol (4 g/kg). CONCLUSIONS: These results indicate that blockade NPY Y5 receptor activity modulates the onset and maintenance of ethanol self-administration. For this reason, NPY-Y5 receptor antagonists may be useful in medical management of alcohol abuse and alcoholism.     

Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.