p53 abnormalities and genomic instability in primary human breast carcinomas.

Abnormalities in the p53 tumor suppressor gene have been shown to affect cell cycle control and lead to genetic instability in cell lines of murine and human origin. We have examined genetic instability in 183 primary human breast carcinomas with and without p53 abnormalities. Mutation analysis was performed by constant denaturant gel electrophoresis and DNA sequencing, and abnormal protein expression was examined by immunohistochemical staining methods. Genetic instability was studied by detection of gene amplification, allelic loss, karyotype analysis, and fluorescent in situ hybridization. We found a significant association between p53 abnormalities and genetic instability detected by these methods.

BRCA2 and p53 mutations in primary breast cancer in relation to genetic instability.

The products of the BRCA breast cancer susceptibility genes have been implicated in cell cycle control and DNA repair. It has been suggested that mutations in the p53 gene are a necessary step in tumorigenesis in BRCA tumors. We tested samples from 402 breast cancer patients for germ-line BRCA2 and p53 mutations in tumors. p53 mutations are more frequent in BRCA2 mutation carriers than they are in controls. Tumors with mutations in either gene had multiple chromosomal abnormalities, as shown by cytogenetic analysis.

Clinical grade expansion of MSCs.

Producing advanced therapy medicinal products (ATMP) according to Good Manufacturing Practice (GMP) guidelines represents a global challenge for the expansion of cells intended for human use. Mesenchymal stromal cells (MSCs) from different sources are one of the most actively developed cell type for a variety of clinical applications in cellular therapy. Complying with GMP means defining accurately both the production process and the release criteria required for a final safe product. We have here reported our manufacturing experience on 103 consecutive clinical-grade in vitro expansions of both bone marrow-derived and umbilical cord-derived mesenchymal stromal cells together with description of methods and reagents utilized in our Cell Factory. The same animal- and serum-free medium, additioned with human platelet lysate, has been used for all the expansions performed. This is the largest experience published so far with this alternative and clinical-grade reagent (compared to the traditional fetal bovine serum) and shows the feasibility and the reproducibility of the method. Indeed, we have been able to produce a sufficient number of MSCs to treat 57 patients so far, enrolled in 7 different experimental phase I/II protocols.

Genomic instability and poor prognosis associated with abnormal TP53 in breast carcinomas. Molecular and immunohistochemical analysis.

Alterations of the TP53 gene were analyzed in samples from 87 primary breast cancer patients, using molecular and immunohistochemical approaches. Mutations were detected in 17% of the samples, using polymerase chain reaction (PCR) and constant denaturant gel electrophoresis (CDGE) on exons 5-8 of the TP53 gene, and were confirmed by sequencing. Abnormal TP53 protein staining was found in 55% of the primary samples, using the monoclonal TP53 antibody DO7. A statistically significant association was found between TP53 mutations and abnormal protein staining (p = 0.002). Our results suggest that dysfunction of the TP53 protein is associated with tumor progression, as we found an association between TP53 abnormalities and accumulation of genetic lesions, measured as overall allelic imbalance (AI), homogeneously staining regions (HSR) and strong ERBB2 overexpression. Furthermore, patients with TP53 mutation had a highly elevated risk of dying from breast cancer during the study period (p < 0.001, RR = 10.68) at a median follow-up time of 42 months. Abnormal TP53 staining was much more frequent than the mutations, but it was not of prognostic significance, whereas strong staining was an independent prognostic factor. We therefore conclude that loss of functional TP53 leads to genetic instability, resulting in poorer short-term prognosis, and that only strong staining of TP53, and not abnormal protein staining in general, is of prognostic significance.

Molecular genetics and cytogenetics of breast carcinomas: comparison of the two methods.

Molecular genetics and cytogenetics are two different approaches to studying genetic changes in breast carcinoma. We have used karyotype analysis, fluorescence in situ hybridization, and molecular analysis of allelic imbalance on chromosomes 7q and 16q and on both arms of chromosome 17, to study 85 breast carcinomas. Twenty-five of these samples gave results that could be used to compare the two methods. Sixty-nine chromosome arms were compared, of which 48 (70%) gave concordant molecular and cytogenetical results. Samples were processed for karyotyping both by harvesting directly from the fresh tissue and after selective culture for a few days. Karyotypes among the direct harvest samples matched significantly better with the molecular genetics results than karyotypes among the cultured cell preparations.

Cloning and characterization of MDDX28, a putative dead-box helicase with mitochondrial and nuclear localization.

A cDNA encoding a novel member of the helicase family, MDDX28, has been cloned from a human testis library. This apparently intronless gene was transcribed in all tissues studied. MDDX28 encodes a protein of 540 amino acids, with approximately 30% homology to other helicases over the core region, containing all the conserved DEAD-box helicase motifs. No homologue is known. MDDX28 has RNA and Mg(2+)-dependent ATPase activity. Subcellular localization studies of MDDX28 using oligoclonal antibodies raised against the protein as well as its enhanced green fluorescence protein (EGFP) demonstrated that the protein is localized in the mitochondria and the nucleus. To our knowledge, MDDX28 is the first member of the RNA helicase described with this dual location. The nuclear localization of MDDX28 depended on active RNA polymerase II transcription, suggesting that the protein could be transported to and from the nucleus. This was confirmed further in an interspecies heterokaryon assay, in which MDDX28 was seen to translocate to the nucleus and mitochondria. The mitochondrial uptake of the MDDX28-EGFP-N1 fusion protein was inhibited by carbonyl cyanide p-(trichloromethoxy)phenylhydrazone. Our results indicate that MDDX28 can be transported between the mitochondria and the nucleus.

TP53 abnormalities and genetic instability in breast cancer.

TP53 abnormalities in breast carcinomas and inherited TP53 changes in breast cancer patients and in Li-Fraumeni-like families were looked for. Tumours were screened for mutations in the TP53 gene by means of the PCR-CDGE method followed by PCR and direct sequencing. Allelic loss was determined by polymorphic markers, by comparing normal and tumour DNA. Abnormal protein expression was examined by immunohistochemical staining. TP53 abnormalities in the tumours were examined in relation to genetic instability, clinical data and family history. Genetic instability was studied by detection of oncogene amplification, allelic loss, karyotype analysis and fluorescent in situ hybridization, FISH. Our studies showed that TP53 abnormalities were significantly associated with amplification of the erbB2 oncogene and allelic loss on chromosome 17. Chromosomal abnormalities were also significantly more common in tumours with TP53 abnormalities. Looking at clinical data we found significant association between TP53 abnormalities and poor prognosis.

Mutations in exons 5-8 of the p53 gene, independent of their type and location, are associated with increased apoptosis and mitosis in invasive breast carcinoma.

In breast cancer, mutations located in the zinc-binding functional domains of the p53 gene have been reported to predict a worse prognosis and a worse response to treatment with doxorubicin, compared with mutations in other parts within exons 5-8 of the gene. Similarly, mutations in residues of p53 that directly contact DNA have been associated with a poor prognosis. To investigate whether these specific p53 mutations are associated with differences in the rate of apoptosis and/or mitosis, or expression of the anti-apoptotic Bcl-2 protein, these parameters were evaluated in 89 invasive breast cancers with a confirmed p53 mutation in exons 5-8 and in 99 tumours without a p53 mutation in exons 5-8. Neither mutations located in the zinc-binding functional domains nor mutations in residues that directly contact DNA were associated with alterations in mitotic or apoptotic activity. However, compared with the wild-type p53 tumours, both apoptotic and mitotic indices showed an approximately two-fold increase in the mutant p53 group ( p< 0. 001). The presence of a p53 mutation was also associated with the presence of tumour necrosis ( p< 0.001), high tumour grade ( p< 0. 001) and low expression of Bcl-2 ( p< 0.001). Our data support the concept that in invasive breast carcinoma, loss of p53 function is involved in enhanced proliferation rather than decreased apoptosis and that the resulting acceleration of cell turnover may enhance clonal evolution and tumour progression.