The mechanisms causing resistance to chemotherapeutic drugs in cancer patients are poorly understood. Recent evidence suggests that different forms of chemotherapy may exert their cytotoxic effects by inducing apoptosis. The tumor suppressor gene P53 has a pivotal role inducing apoptosis in response to cellular damage. In vitro investigations have shown intact p53 to play a critical role executing cell death in response to treatment with cytotoxic drugs like 5-fluorouracil, etoposide and doxorubicin. Recently, mutations in the P53 gene were found to confer resistance to anthracyclines in a mouse sarcoma tumor model, and overexpression of the p53 protein (which, in most cases, is due to a mutated gene) was found to be associated with lack of response to cisplatin-based chemotherapy in non-small cell lung cancer. Previous studies have shown mutations in the P53 gene or overexpression of the p53 protein to predict a poor prognosis, but also a beneficial effect of adjuvant radiotherapy or chemotherapy in breast cancer. In this study we present data linking specific mutations in the P53 gene to primary resistance to doxorubicin therapy and early relapse in breast cancer patients.

At present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed "constant denaturant gel electrophoresis" (CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss of heterozygosity for a chromosome 17p marker. By immunostalning techniques, only 6 of the 32 tumors had elevated p53 expression. However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or pl44D6 markers did not associate with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE is a rapid and effective technique to screen for p53 mutations.A wide variety of human tumors have been shown to be associated with changes in the p53 gene (1-8). As the list of cancers with p53 mutations increases, there is a need for an accurate and rapid screening technique for these mutations. Unlike some of the dominant oncogenes, p53 can be inactivated by a diverse set of mutations scattered through several important regions of the gene (7). Many recent surveys of p53 mutations have used loss of heterozygosity (LOH) at polymorphic markers closely linked to p53 as an indirect assay for p53 inactivation (9-11). While such a screen is rapid, it cannot detect point mutations, and its accuracy is limited, since some of the nearby polymorphic markers might undergo deletions that do not extend into the p53 locus. Alternatively, because mutations in the p53 gene frequently result in a mutant p53 protein that is significantly overexpressed compared with the low levels of the wild-type protein, other p53 surveys have used immunostaining techniques to screen for mutations (12)(13)(14). Unfortunately, the p53 mutations that result in either a lack of protein or the same low levels of p53 as are present in cells containing the wild-type p53 cannot be detected by this method. The most sensitive screening technique is to sequence the genomic region encoding p53. Although sequencing is quite sensitive, it is also laborintensive. There are several new nucleic acid-based screening methods that can rapidly detect mutations within short fragments of DNA. These techniques include RNase protection assays (15), single-strand conformational polymorphisms (SSCP) (16, 17), denaturing gradient gel electrophoresis (DGGE) (18,19), and detection of base-pair mismatches with hydroxylamine and osmiu...

We identified germline p53 mutations in seven of 235 (3.0%) children with osteosarcoma. Four of these mutations were found in patients who did not have first-degree relatives with cancer. Although genetic transmission of the altered p53 gene could not be tested in this survey because of how it was designed, it is possible that predictive testing for p53 mutations could identify unaffected relatives of gene carriers who also have a high risk for the development of cancer. This study provides evidence for the importance of considering children with osteosarcoma for predictive testing for germline p53 mutations.

Background: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.

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