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M Perucho

Publications and source records attributed to M Perucho.

At least 19 recordsLinked to original sources
PubMed2001

Identification of novel deletion regions on chromosome arms 2q and 6p in breast carcinomas by amplotype analysis.

DNA fingerprinting by arbitrarily primed PCR (AP-PCR) was employed to identify molecular genetic alterations in 37 primary breast carcinomas. AP-PCR is a PCR-based technique that uses only one primer of arbitrary sequence that generates a molecular karyotype (amplotype) of tumors. The breast cancer amplotype generated with two arbitrary primers (MCG1 and Blue) showed a relatively high frequency (more than 20% of the tumors) of gains at chromosomes 1, 4, and 8, and of losses at chromosomes 2, 4, 6, 9, 10, 11, 13, and the X chromosome. We further analyzed the regions most commonly gained at chromosome 8 (47%) and lost at chromosomes 2 (38%) and 6 (49%) by determining the subchromosomal localization of the fingerprint bands from these chromosomes. The region of gain at chromosome 8 was mapped at 8q24.1, close to MYC. Band MCG1-A1 was assigned to chromosome band 2q22, and band Blue-J was assigned to 6p21. Common losses of these chromosomal regions have not been described for breast cancer. To map these deletion regions more precisely, we performed loss of heterozygosity (LOH) analysis by microallelotyping on 20 of the 37 cancers previously analyzed by AP-PCR and another additional 52 breast carcinomas. The results suggest that the regions at 2q21-24 and 6p21-23 may harbor novel tumor suppressor genes for breast cancer. LOH at 2q21-24 (D2S2304) was more frequent in high-grade tumors (59%) than in low-grade tumors (29%) (P = 0.03). This suggests that this genetic alteration may be associated with tumor progression and shows the power of the amplotype approach in detecting novel genetic alterations that are useful as clinical parameters of breast cancer.

Breast Neoplasms↗
PubMed2000

Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution.

A remarkable instability at simple repeated sequences characterizes gastrointestinal cancer of the microsatellite mutator phenotype (MMP). Mutations in the DNA mismatch repair gene family underlie the MMP, a landmark for hereditary nonpolyposis colorectal cancer. These tumors define a distinctive pathway for carcinogenesis because they display a particular spectrum of mutated cancer genes containing target repeats for mismatch repair deficiency. One such gene is BAX, a proapoptotic member of the Bcl-2 family of proteins, which plays a key role in programmed cell death. More than half of colon and gastric cancers of the MMP contain BAX frameshifts in a (G)(8) mononucleotide tract. However, the functional significance of these mutations in tumor progression has not been established. Here we show that inactivation of the wild-type BAX allele by de novo frameshift mutations confers a strong advantage during tumor clonal evolution. Tumor subclones with only mutant alleles frequently appeared after inoculation into nude mice of single-cell clones of colon tumor cell lines with normal alleles. In contrast, no clones of BAX-expressing cells were found after inoculation of homozygous cell clones without wild-type BAX. These results support the interpretation that BAX inactivation contributes to tumor progression by providing a survival advantage. In this context, survival analyses show that BAX mutations are indicators of poor prognosis for both colon and gastric cancer of the MMP.

Animals↗
PubMed2000

Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability.

Many lines of evidence suggest that the retinoblastoma protein interacting zinc finger gene RIZ is a strong candidate for the tumor suppressor locus on 1p36, a region commonly deleted in many human cancers with chromosomal instability. In addition, a role for RIZ in tumors of the microsatellite instability pathway is suggested by frequent frameshift mutations in hereditary non-polyposis colorectal carcinomas. Here we studied RIZ mutations in sporadic cancers with microsatellite instability. Frameshift mutations in the two coding polyadenosine tracks of RIZ were found in 19 (48%) of 40 gastric carcinomas, 6 (33%) of 18 endometrial carcinomas, 14 (26%) of 51 of colorectal carcinomas, and 7 (54%) of 13 cell lines. Eleven tumor tissues showed biallelic inactivation of RIZ. In contrast, no frameshift mutations were found in 70 microsatellite stable tumors. These results suggest an important role for RIZ in sporadic cancers with microsatellite instability.

Alleles↗
PubMed2000

Frequent chromosome 8q gains in human small cell lung carcinoma detected by arbitrarily primed-PCR genomic fingerprinting.

The arbitrarily primed-polymerase chain reaction (AP-PCR) genomic fingerprinting method was applied to detect chromosomal numerical imbalances in 13 small cell lung carcinomas (SCLC). Increases and decreases in the intensity of the AP-PCR bands from several chromosomes, representing gains of chromosomes 1, 7, 16, and X, and losses of chromosomes 2, 10, and 22, were recurrent events in independent tumors. In addition, the most common alterations detected were increases in signal intensity of three AP-PCR bands derived from genomic sequences on chromosome 8q (eight of 13 tumors: 62%). To define whether the 8q gains observed in the AP-PCR analysis include the C-MYC gene at chromosome 8q24 or not, we performed targeted AP-PCR analysis of the C-MYC gene. The C-MYC gene was amplified in five of the eight tumors with gains of 8q, but in none of the remaining five tumors in which 8q gains were not detected. These results indicate that chromosome 8q gain occurs frequently in SCLC and gained regions often, but do not always, include the C-MYC gene. Moderate increases in copy number of the C-MYC gene and additional gene(s) in the 8q region appear to be under positive selection during SCLC progression.

Carcinoma, Small Cell↗
PubMed2000

Amplotyping of microdissected, methanol-fixed lung carcinoma by arbitrarily primed polymerase chain reaction.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to detect somatic genetic alterations in lung carcinomas. DNA fingerprints generated by a single arbitrary primer were compared between normal and tumor tissues of the same individuals. We adapted the technique to the use of tissue fixed with methanol, which allowed the analysis of small areas of tissue by microdissection. This improvement of the fingerprinting technique permitted the study of tumors at early stages of progression. Loss of sequences from chromosome 7 was detected in 41.7% of adenocarcinomas and from chromosome 22 in 84.6% of small-cell carcinomas. Gains of sequences from chromosomes 1, 8 and 13 were detected in more than 40% of adenocarcinomas and in chromosome 2 in 63.3% of squamous-cell carcinomas. Our results indicate that allelic imbalances at these chromosomal regions are common genetic abnormalities in lung carcinomas. Loss of sequences from chromosome 22q13.3, found in 11 of 13 small-cell carcinomas, were confirmed by microsatellite PCR analysis. We show that the use of our improved AP-PCR fingerprinting permits the detection of both losses and gains of novel chromosomal regions early during lung cancer development. Our results indicate that early-stage tumors tend to have more allelic imbalances than relatively advanced tumors, suggesting a high tumor genetic heterogeneity in the early stages of lung tumor progression.

Adenocarcinoma↗
PubMed1999

Frameshift mutations at mononucleotide repeats in caspase-5 and other target genes in endometrial and gastrointestinal cancer of the microsatellite mutator phenotype.

The majority of tumors from hereditary nonpolyposis colorectal cancer families and a subset of unselected gastrointestinal and endometrial tumors exhibit a microsatellite mutator phenotype (MMP) that leads to the accumulation of hundreds of thousands of clonal mutations in simple repeat sequences. The mutated genes with positive or negative roles in cell growth or survival in aneuploid gastrointestinal cancer (e.g., APC, K-ras, and p53) are less frequently mutated in near-diploid MMP gastrointestinal tumors. These tumors accumulate mutations in other genes, such as DNA mismatch repair hMSH3 and hMSH6, transforming growth factor-beta type II receptor, and BAX. All these genes carry, within their coding sequences, mononucleotide repeats that are preferred targets for the MMP. Endometrial carcinoma is the most common type of extracolonic neoplasia in the hereditary nonpolyposis colorectal cancer syndrome, but the spectrum of its target cancer genes is not well characterized. Here, we report that endometrial cancer of the MMP also accumulates mutations in genes that are typically mutated in gastrointestinal cancer of the mutator pathway, including BAX (55%), hMSH3 (28%), and hMSH6 (17%). We also report the detection of frameshift mutations in caspase-5, a member of the caspase family of proteases that has an (A)10 repeat within its coding region, in MMP tumors of the endometrium, colon, and stomach (28, 62, and 44%, respectively). We therefore suggest caspase-5 as a new target gene in the microsatellite mutator pathway for cancer.

Caspases↗
PubMed1999

Impairment of the proapoptotic activity of Bax by missense mutations found in gastrointestinal cancers.

We have reported previously that codon 169 of the proapoptotic gene BAX is a mutational hot spot in gastrointestinal cancer. Two different mutations were found in this codon, replacing the wild-type threonine by alanine or methionine. To compare the proapoptotic activity of these Bax mutants with wild-type Bax, we established an ecdysone (muristerone A)-inducible system in cultured human embryonal kidney 293 cells. Addition of muristerone A induced a dose-dependent decrease in the viability of cells transfected with wild-type BAX, but this loss of viability was inhibited in cells transfected with BAX mutants. Furthermore, muristerone A induced morphological changes characteristic of apoptosis, including cell shrinkage, rounding, formation of apoptotic bodies, detachment and nuclear condensation and fragmentation, in cells transfected with wild-type BAX. These hallmarks of apoptosis were clearly diminished in cells transfected with BAX mutants. Mutation of threonine 169 did not affect the binding of Bax to Bax, Bcl-2, or Bcl-X(L). These results demonstrate that missense mutations at codon 169 of BAX are functional because they inhibit its apoptotic activity. This is the first report of the functional significance of missense mutations in BAX, or any other proapoptotic member of the Bcl-2 family, in primary human tumors.

Alanine↗
PubMed1999

Differential contribution of the ERK and JNK mitogen-activated protein kinase cascades to Ras transformation of HT1080 fibrosarcoma and DLD-1 colon carcinoma cells.

Although an important contribution of ERK and JNK mitogen-activated protein kinase (MAPK) activation in Ras transformation of rodent fibroblasts has been determined, their role in mediating oncogenic Ras transformation of human tumor cells remains to be established. We have utilized the human HT1080 fibrosarcoma and DLD-1 colon carcinoma cell lines, which contain endogenous mutated and oncogenic N- and K-ras alleles, respectively, to address this role. Study of these cells is advantageous over Ras-transformed rodent model cell systems for two key reasons. First, the ras mutations occurred naturally in the progression of the tumors from which the cell lines were derived, rather than due to overexpression of an exogenously introduced gene. Second, although these tumor cells possess defects in multiple genetic loci, it has been established that mutated Ras contributes significantly to the transformed phenotype of these cells. Clonal variant lines of HT1080 and DLD-1 have been isolated which have lost the oncogenic ras allele and exhibit a corresponding impairment in growth transformation in vitro and in vivo. We found that upregulation of Raf/MEK/ERK and JNK correlated with expression of oncogenic Ras in HT1080, but not DLD-1 cells. Furthermore, inhibition of ERK activation in parental HT1080 cells caused the same changes in cell morphology and actin stress fiber organization seen with loss of expression of activated N-Ras(61K). Thus, we suggest that constitutive activation of the Raf/MEK/ERK and JNK pathways is necessary for Ras-induced transformation of HT1080 but not DLD-1 cells. These results emphasize that cell type differences exist in the signaling pathways by which oncogenic Ras causes transformation.

3T3 Cells↗
PubMed1999

Gastric cancers of the microsatellite mutator phenotype display characteristic genetic and clinical features.

BACKGROUND & AIMS: Colon cancer of the microsatellite mutator phenotype (MMP) exhibits significant genotype differences from cancer without the MMP. Twenty-nine MMP-positive gastric cancers were analyzed to clarify if these genotype differences were also associated with distinctive clinicopathologic features. METHODS: Alterations of p53, beta2-microglobulin (beta2M ), hMLH1, and hMSH2 genes were analyzed by using polymerase chain reaction, single-strand conformational polymorphism, sequencing, microallelotyping, hypermethylation assays, and immunostaining. The results were contrasted with mutations in BAX, hMSH3, and hMSH6, genes target for the MMP. RESULTS: Tumors with the MMP had a significantly lower incidence of p53 gene mutations than the other tumors and often contained beta2M gene somatic mutations. Many tumors contained concomitant genetic and epigenetic alterations in DNA mismatch repair genes, hMLH1, hMSH2, hMSH3, and hMSH6. Gastric cancer of the MMP was associated with well/moderate differentiation, distal location, and better survival. CONCLUSIONS: Analysis of somatic alterations in microsatellite sequences and in cancer genes target for the MMP is useful for the classification of groups of gastric cancers with different prognosis. The results further support the concept that (gastric) cancer of the MMP represents a distinctive oncogenic pathway because the mutated cancer genes are usually different from those found in tumors without the MMP.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Prognostic significance of apoptosis regulators in breast cancer.

Dysregulation of normal programmed cell death mechanisms plays an important role in the pathogenesis and progression of breast cancer, as well as in responses of tumors to therapeutic intervention. Overexpression of anti-apoptotic members of the Bcl-2 family such as Bcl-2 and Bcl-X(L) has been implicated in cancer chemoresistance, whereas high levels of pro-apoptotic proteins such as Bax promote apoptosis and sensitize tumor cells to various anticancer therapies. Though the mechanisms by which Bcl-2 family proteins regulate apoptosis are diverse, ultimately they govern decision steps that determine whether certain caspase family cell death proteases remain quiescent or become active. To date, approximately 17 cellular homologs of Bcl-2 and at least 15 caspases have been identified in mammals. Other types of proteins may also modulate apoptotic responses through effects on apoptosis-regulatory proteins, such as BAG-1-a heat shock protein 70 kDa (Hsp70/Hsc70)-binding protein that can modulate stress responses and alter the functions of a variety of proteins involved in cell death and division. In this report, we summarize our attempts thus far to explore the expression of several Bcl-2 family proteins, caspase-3, and BAG-1 in primary breast cancer specimens and breast cancer cell lines. Moreover, we describe some of our preliminary observations concerning the prognostic significance of these apoptosis regulatory proteins in breast cancer patients, contrasting results derived from women with localized disease (with or without node involvement) and metastatic cancer.

Antineoplastic Agents↗
PubMed1998

Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: allelic losses in chromosome 10q in lung cancers.

DNA fingerprinting using arbitrarily primed PCR (AP-PCR) is useful for detecting cancer-specific DNA aberrations without targeting any particular genes or knowing any nucleotide sequences in advance. AP-PCR fingerprinting is an efficient method for finding loss of anonymous chromosomal regions in cancers. We analyzed DNA from 44 human non-small cell lung cancers by fingerprinting using a single primer and found a loss of signal intensity in a DNA fragment amplified from chromosome 10 (fragment F) in 15 tumors. The detailed location of the fragment F locus on chromosome 10q was determined by PCR-based analysis of radiation hybrid panels using a sequence-tagged site established for the fragment. In 12 of the 15 tumors, loss of the signal detected by AP-PCR fingerprinting was in agreement with the results obtained by analysis of allelic imbalances using 7 polymorphic CA-microsatellite DNA markers for loci around the fragment F locus (p=0.0009). We conclude that a hitherto unknown suppressor gene for lung cancer resides at 10q in the vicinity of fragment F.

Alleles↗
PubMed1998

Molecular karyotype (amplotype) of metastatic colorectal cancer by unbiased arbitrarily primed PCR DNA fingerprinting.

Genomic instability characterizes the aneuploid cancer cell. Losses of genetic material are critical in cancer by exposing recessive mutations in tumor suppressor genes. Gains of genetic material also may lead to overexpression of genes contributing to tumor progression either in the presence or absence of mutation. However, the detection of moderate gains (such as tri-tetraploidy) has been a challenge in cancer research. Unbiased DNA fingerprinting by the arbitrarily primed PCR allows the detection of moderate gains (in addition to losses) of DNA sequences of known chromosomal localization. We have generated in this manner a molecular karyotype of metastatic colon cancer. This amplotype shows that sequences from several chromosomes undergo both losses (1, 4, 9, 14, and 18) and gains (6, 7, 12, and 20) in over half of the tumors. Moreover, gains of sequences from chromosomes 8 and 13 occurred in most tumors, indicating the existence in these chromosomes of positive regulators of cell growth or survival that are under strong positive selection during tumor progression. We conclude that overrepresentation of these chromosomal regions is a critical step for metastatic colorectal cancer. Comparative amplotype analysis from primary and metastatic tumors suggest the existence in chromosome 4 of gene(s) whose loss is specifically selected in cells that reach the metastatic stage.

Alleles↗