Mutation analysis of p53, K-ras, and BRAF genes in colorectal cancer progression

Gene mutations in APC, K-ras, and p53 are thought to be essential events for colorectal cancer development. Recent data seem to indicate that K-ras and p53 mutations rarely co-exist in the same tumor, indicating that these alterations do not represent a synergistic evolutionary pathway. Moreover, an inverse relation between K-ras gene activation and BRAF mutations has been demonstrated, suggesting alternative pathways for colorectal cancer transformation. To reconstruct the chronological modulation of these gene mutations during cell transformation and colorectal cancer progression, mutations of p53, K-ras, and BRAF genes were analyzed by Single Strand Conformation Polymorphism (SSCP) or sequencing analysis in 100 colorectal cancer samples, evenly distributed among different Dukes' stages. We found mutations in p53, K-ras, and BRAF genes in 35%, 30%, and 4% of tumors, respectively, and observed a minimal or no co-presence of these gene alterations. Moreover, the frequency of molecular p53 mutations increased as tumor stage increased, suggesting an important role for this gene in the progression of colorectal cancer. Conversely, K-ras or BRAF genes were not related to tumor stage or location. These data seem to indicate the absence of a co-presence of the genes, highlighting the possibility of multiple pathways for colorectal tumor progression. Moreover, mutations in p53, K-ras, and BRAF are not present in about one-third of colorectal cancers and therefore other gene mutations need to be investigated to better understand molecular mechanisms at the basis of cell transformation and the progression of colorectal cancer.     

Germline p53 single-base changes associated with Balkan endemic nephropathy

The Balkan endemic nephropathy (BEN) is a significant clinical and scientific problem in need of novel effective therapies. Though many genetic and environmental factors have been investigated the basis, cause, and predisposition to BEN are still unclear. In this study, based on the hypothesis that the genetic pathways leading to BEN might be associated with p53 dysfunction, we screened for p53 gene mutations 90 Bulgarian BEN patients using optimized PCR-SSCP-sequencing analysis. Germline p53 single-base changes were found in blood samples in 10% of BEN cases. Three of them caused amino acid substitutions (p.Arg283Cys, p.Gln317His, and p.Lys321Glu); the other six were either synonymous amino acid substitutions (p.Arg213Arg) or intron polymorphisms (T14766C). To the best of our knowledge, these are the first data investigating tumor suppressor gene mutations in patients with BEN. The obtained results are in support of our hypothesis that p53 gene alterations are possibly involved in BEN genetic pathways.     

Binding of MAR-DNA elements by mutant p53: Possible implications for its oncogenic functions

The tumor suppressor p53 is a multifunctional protein whose main duty is to preserve the integrety of the genome. This function of wild-type p53 as “guardian of the genome” is achieved at different levels, as a cell cycle checkpoint protein, halting the cell cycle upon DNA damage, and via a direct involvement in processes of DNA repair. Alternatively, p53 can induce apoptosis. Mutations in the p53 gene occur in about 50% of all human tumors and eliminate the tumor suppressor functions of p53. However, many mutant p53 proteins have not simply lost tumor suppressor functions but have gained oncogenic properties which contribute to the progression of tumor cells to a more malignant phenotype. The molecular basis for this gain of function of mutant p53 is still unknown. However, mutant (mut) p53 specifically binds to nuclear matrix attachment region (MAR) DNA elements. MAR elements constitute important higher order regulatory elements of chromatin structure and function. By binding to these elements, mut p53 could modulate important cellular processes, like gene expression, replication, and recombination, resulting in phenotypic alterations of the tumor cells. Mut p53 thus could be the first representative of a new class of oncogenes, which exert their functions via long-range alterations or perturbation of chromatin structure and function. © 1996 Wiley-Liss, Inc.     

The promise and obstacle of p53 as a cancer therapeutic agent

p53 is a tumor suppressor gene that is mutated in greater than 50% of human cancers. The action of p53 as a tumor suppressor involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Loss of p53 function therefore allows the uncontrolled proliferation associated with cancerous cells. While design of most anti-cancer agents has focused on targeting and inactivating cancer promoting targets, such as oncogenes, recent attention has been given to restoring the lost activity of tumor suppressor genes. Because the loss of p53 function is so prevalent in human cancer, this protein is an ideal candidate for such therapy. Several gene therapeutic strategies have been employed in the attempt to restore p53 function to cancerous cells. These approaches include introduction of wild-type p53 into cells with mutant p53; the use of small molecules to stabilize mutant p53 in a wild-type, active conformation; and the introduction of agents to prevent degradation of p53 by proteins that normally target it. In addition, because mutant p53 has oncogenic gain of function activity, several approaches have been investigated to selectively target and kill cells harboring mutant p53. These include the introduction of mutant viruses that cause cell death only in cells with mutant p53 and the introduction of a gene that, in the absence of functional p53, produces a toxic product. Many obstacles remain to optimize these strategies for use in humans, but, despite these, restoration of p53 function is a promising anti-cancer therapeutic approach.     

p33(ING1) enhances UVB-induced apoptosis in melanoma cells

The biological functions of the tumor suppressor ING1 have been studied extensively in the past few years since it was cloned. It shares many biological functions with p53 and has been reported to mediate growth arrest, senescence, apoptosis, anchorage-dependent growth, chemosensitivity, and DNA repair. Some of these functions, such as cell cycle arrest and apoptosis, have been shown to be dependent on the activity of both ING1 and p53 proteins. Two recent reports by Scott and colleagues demonstrate that p33(ING1) (one of the ING1 isoforms) translocates to the nucleus and binds to PCNA upon UV irradiation. Here we report that p33(ING1) mediates UV-induced cell death in melanoma cells. We found that overexpression of p33(ING1) increased while the introduction of an antisense p33(ING1) plasmid reduced the apoptosis rate in melanoma cells after UVB irradiation. We also demonstrated that enhancement of UV-induced apoptosis by p33(ING1) required the presence of p53. Moreover, we found that p33(ING1) enhanced the expression of endogenous Bax and altered the mitochondrial membrane potential. Taken together, these observations strongly suggest that p33(ING1) cooperates with p53 in UVB-induced apoptosis via the mitochondrial cell death pathway in melanoma cells.     

Abrogation of p53 by its antisense in MCF-7 breast carcinoma cells increases cyclin D1 via activation of Akt and promotion of cell proliferation

The p53 protein has been a subject of intense research interest since its discovery as about 50% of human cancers carry p53 mutations. Mutations in the p53 gene are the most frequent genetic lesions in breast cancers suggesting a critical role of p53 in breast cancer development, growth and chemosensitivity. This report describes the derivation and characterization of MCF-7As53, an isogenic cell line derived from MCF-7 breast carcinoma cells in which p53 was abrogated by antisense p53 cDNA. Similar to MCF-7 and simultaneously selected hygromycin resistant MCF-7H cells, MCF-7As53 cells have consistent basal epithelial phenotype, morphology, and estrogen receptor expression levels at normal growth conditions. Present work documents investigation of molecular variations, growth kinetics, and cell cycle related studies in relation to absence of wild-type p53 protein and its transactivation potential as well. Even though wild-type tumor suppressor p53 is an activator of cell growth arrest and apoptosis-mediator genes such as p21, Bax, and GADD45 in MCF-7As53 cells, no alterations in expression levels of these genes were detected. The doubling time of these cells decreased due to depletion of G0/G1 cell phase because of constitutive activation of Akt and increase in cyclin D1 protein levels. This proliferative property was abrogated by wortmannin, an inhibitor of PI3-K/Akt signaling pathway. Therefore this p53 null cell line indicates that p53 is an indispensable component of cellular signaling system which is regulated by caveolin-1 expression, involving Akt activation and increase in cyclin D1, thereby promoting proliferation of breast cancer cells.     

An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis

Simian virus 40 large T antigen transforms cells by sequestration and inactivation of the tumor suppressor proteins p53, retinoblastoma gene product (pRb), and the pRb-related proteins p107 and p130. Thus, the absence of functional p53 is expected to promote T antigen-mediated tumorigenesis. However, in a transgenic mouse model of T antigen-mediated beta cell carcinogenesis (Rip1Tag2), tumor volumes are significantly diminished when these mice are intercrossed with p53-deficient mice. Whereas the incidence of beta tumor cell apoptosis is unaffected, their proliferation rate is reduced in p53-deficient beta cell tumors in vivo and in cell lines established from these tumors in vitro. Biochemical analyses reveal higher levels of T antigen in wild-type tumor cells as compared to p53-deficient tumor cells. The data indicate that p53 stabilizes SV40 large T antigen, thereby augmenting its oncogenic potential as manifested by increased proliferation rates in wild-type beta tumor cells as compared to p53-deficient beta tumor cells.     

The use of p53 as a tool for human cancer therapy

The p53 tumor suppressor is a central component of a system that reinforces genetic stability of somatic cells in animals and humans. Inactivation of this gene occurs virtually in every cancer case, which eliminates results in further rapid accumulation of additional mutations leading to progression of a cancer cell toward more malignant phenotype. The mechanisms of p53 inhibition in cancer include point mutations leading to accumulation of inactive protein, deletion of the whole gene, or its portion, alteration in the genes involved in regulation of activity of p53, and defects in the genes controlled by p53. In addition, oncogenic viruses encode specialized proteins that are entitled to modify p53 functions in order to provide optimal condition for replication of viral genome. These viral proteins play central role in viral carcinogenesis, including 95% of cases of cervical carcinoma in women. The approacheas to restoration of p53 activity depend on particular type of alteration within the p53 pathway. In some cases an effective mean would be introduction of exogenous p53, particularly with the use of adenoviral vectors. There are also approaches in development that target reactivation of mutant proteins, or suppress natural inhibitors of p53. The review summarizes various schemes for therapy and prevention of cancer that are based on our knowledge of the p53 gene functions. Potential usefulness of the approaches for practical applications is discussed.     

Genomic deletion and promoter methylation status of Hypermethylated in Cancer 1 (HIC1) in mantle cell lymphoma

Mantle cell lymphomas (MCL), characterized by the t(11;14)(q13;q32), frequently carry secondary genetic alterations such as deletions in chromosome 17p involving the TP53 locus. Given that the association between TP53-deletions and concurrent mutations of the remaining allele is weak and based on our recent report that the Hypermethylated in Cancer 1 (HIC1) gene, that is located telomeric to the TP53 gene, may be targeted by deletions in 17p in diffuse large B-cell lymphoma (DLBCL), we investigated whether HIC1 inactivations might also occur in MCL. Monoallelic deletions of the TP53 locus were detected in 18 out of 59 MCL (31%), while overexpression of p53 protein occurred in only 8 out of 18 of these MCL (44%). In TP53-deleted MCL, the HIC1 gene locus was co-deleted in 11 out of 18 cases (61%). However, neither TP53 nor HIC1 deletions did affect survival of MCL patients. In most analyzed cases, no hypermethylation of the HIC1 exon 1A promoter was observed (17 out of 20, 85%). However, in MCL cell lines without HIC1-hypermethylation, the mRNA expression levels of HIC1 were nevertheless significantly reduced, when compared to reactive lymph node specimens, pointing to the occurrence of mechanisms other than epigenetic or genetic events for the inactivation of HIC1 in this entity.     

Use of p53 for therapy of human cancer

Tumor suppressor p53 is the central component of a system maintaining the genetic stability of animal and human somatic cells. Its gene is inactivated in almost all human cancers, allowing a tumor cell to rapidly accumulate additional mutations and progress toward a more malignant phenotype. Yet tumor cells are most sensitive to the suppressor effect of p53 when its function is restored. Hence, restoration of the p53 function is an appealing strategy for anticancer therapy. Various mechanisms inactivate p53 in cancer, including point mutations resulting in synthesis of an inactive mutant protein, deletion of the total gene or its portion, damage to the genes involved in regulating the p53 activity, and defects in p53 target genes. In addition, oncogenic viruses code for the specialized proteins that modify the p53 function to ensure optimal replication of the virus genome. These viral proteins are crucial for virus-induced carcinogenesis, in particular, in 95% of cervical carcinoma cases in women. The approaches to p53 activity restoration depend to a great extent on the defect in p53-dependent signaling. Introduction of exogenous p53 is effective in some cases and is usually achieved with adenoviral vectors. The approaches under study are aimed at restoring the activity of mutant p53 or suppressing the viral inhibitors of p53. The review considers various schemes involving p53 in cancer therapy and prevention and discusses their potential efficacy and prospects of their clinical use.     

Expression of wild-type p53 is not compatible with continued growth of p53-negative tumor cells.

Inactivation of the cellular p53 gene is a common feature of Friend virus-induced murine erythroleukemia cell lines and may represent a necessary step in the progression of this disease. As well, frequent loss or mutation of p53 alleles in diverse human tumors is consistent with the view of p53 as a tumor suppressor gene. To examine the significance of p53 gene inactivation in tumorigenesis, we have attempted to express transfected wild-type p53 in three p53-negative tumor cell lines: murine DP16-1 Friend erythroleukemia cells, human K562 cells, and SKOV-3 cells. We found that aberrant p53 proteins, which differ from wild-type p53 by a single amino acid substitution, were expressed stably in these cells, whereas wild-type p53 expression was not tolerated. The inability of p53-negative tumor cell lines to support long-term expression of wild-type p53 protein is consistent with the view that p53 is a tumor suppressor gene.     

靶向突变p53蛋白的抗肿瘤药物

p53蛋白是人体内十分重要的肿瘤抑制因子,通过调节细胞周期阻滞、诱导细胞凋亡等作用发挥肿瘤抑制功能。突变后的p53蛋白不仅具有显性负性效应（dominant negative effect,DN）抑制野生型p53蛋白功能,而且还通过功能获得性效应（gain of function,GOF）调节细胞代谢、侵袭、迁移等方式促进肿瘤的发生。p53蛋白在超过50%的肿瘤组织中发生突变,是肿瘤细胞区别于正常细胞的一个特异性药物靶点。因此,针对突变p53蛋白开发新型抗癌药物一直是研究的热点。长期以来,由于突变p53蛋白表面较为光滑,缺乏药物结合口袋,使其被认为是一个不可成药的靶点。随着高通量筛选技术的发展以及对突变p53蛋白结构的深入了解,许多靶向突变p53蛋白的小分子化合物被报道并在体外展现出较好的抗肿瘤活性,多款基于突变p53蛋白研发的化合物已经进入临床试验阶段。本文就靶向p53蛋白治疗肿瘤的直接和间接策略进行综述,重点针对突变p53蛋白重激活剂与降解突变p53蛋白的小分子化合物作用机制进行梳理,以期为后续开发靶向突变p53蛋白药物的创新提供帮助。     

突变体p53研究进展

抑癌基因突变是癌症发生过程中一个极为关键的事件。p53作为体内最重要的抑癌基因之一, 在人类癌症中发生突变的频率高达50%。同时, p53突变也是人类遗传病Li-Fraumeni综合征的主要病因。p53最常见的突变形式是错义突变, 所形成的突变体p53不但失去了野生型p53的抑癌功能, 而且还获得了一系列类似于癌基因的功能, 促进了肿瘤的进程。文章拟对突变体p53的结构功能改变, 获得癌基因活性的分子机制, 以及近年来对封闭突变体p53活性所进行的探索等研究方向所取得的进展做一综述。     

Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction

We examined ultraviolet (UV) irradiation and cisplatin treatment damage formation and repair efficiency in the p53 tumor suppressor gene of various cultured cell lines and lymphocytes using a nonradioactive multiplex long quantitative polymerase chain reaction (QPCR) assay, which amplified a 7-kb fragment of the target gene and a 500-bp fragment of the template control to successfully increase the sensitivity and reliability of the assay. The multiplex long QPCR detected a lesion frequency of 0.63 lesions/10kb/10J/m(2) in the p53 gene of fibroblast cells. In addition, the multiplex long QPCR assay detected pronounced differences in the repair of UV damage in the p53 gene among repair-proficient CRL-1475 cells and repair-deficient XP-A and XP-C cells. The multiplex long QPCR assay was also evaluated as a sensitive assay for the detection of DNA damage induced by cisplatin. The data indicated that the lesion frequency in the p53 gene was 1.27-1.75 times higher in the H23 cisplatin-sensitive cell than in the H1435 cisplatin-resistant cell at the IC(70) dose. After 8-h and 24-h repair periods, only 13 and 75% of cisplatin-induced damage had been removed in the H23 cells, whereas these values were 92 and 100% in the H1435 cells. In addition, our data indicate that multiplex long QPCR is a sensitive method for validly estimating repair in freshly isolated lymphocytes. The results suggest that the current protocol of the multiplex long QPCR method can be used to assess the damage formation and repair efficiency of various agents at biologically relevant doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in epidemiological studies.     

Clinical implication of p53 mutation in lung cancer

Lung cancer development involves multiple genetic abnormalities leading to malignant transformation of the bronchial epithelial cells, followed by invasion and metastasis. One of the most common changes is mutation of the p53 tumor suppressor gene. The frequency of p53 alterations in lung cancer is highest in small cell and squamous cell carcinomas. A genetic “signature” of the type of p53 mutations has been associated with carcinogens in cigarette smoke. The majority of clinical studies suggest that lung cancers with p53 alterations carry a worse prognosis, and may be relatively more resistant to chemotherapy and radiation. An understanding of the role of p53 in human lung cancer may lead to more rational targeted approaches for treating this disease. P53 gene replacement is currently under clinical investigation but clearly more effective means of gene deliver to the tumor cells are required. Novel approaches to lung cancer therapy are needed to improve the observed poor patient survival despite current therapies.     

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

Cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.     

A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes.