Katerina Angelopoulou

Circulating antibodies against p53 protein in patients with ovarian carcinoma

Genetic alterations of the p53 tumor suppressor protein are the most frequent molecular events in human carcinogenesis. For as yet unknown reasons, mutant p53 often acts as an immunogen for autoantibody generation. These autoantibodies can be detected in the serum of cancer patients. The presence of such antibodies has been identified in a subset of patients with ovarian carcinoma, but their clinical significance has not been investigated.

Humoral immune response against p53 protein in patients with colorectal carcinoma

p53Aberrations are frequent in colorectal carcinogenesis (40–70%). Because p53 gene mutations typically result in increased p53 protein concentration in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, serum p53 antibodies were quantitatively analyzed in 229 patients with colorectal cancer, using an immunofluorometric procedure. Circulating antibodies against p53 were found in 23% (53/229) of the patients. We quantified antibody concentrations in all positive sera and found that they varied from 300 to 500,000 arbitrary units/l. Sequential analysis of positive sera from 3 patients showed that p53 antibody concentrations change during the course of the disease, reflecting progression or regression. No association was found between the presence of p53 antibodies and age, sex, stage, histological grade and patient relapse‐free or overall survival. These data demonstrate that antibody generation against the p53 tumor‐suppressor protein is a relatively common event in colorectal cancer and that serological analysis for p53 antibodies may have some value for patient monitoring. The test has no value for prognosis.

p53 gene mutation, tumor p53 protein overexpression, and serum p53 autoantibody generation in patients with breast cancer.

OBJECTIVES Autoantibodies against the p53 tumor suppressor protein have been detected in the serum of a proportion of patients with various cancers. The generation of such antibodies has been proposed to be due to either tumor p53 protein accumulation or to the type of p53 gene mutation. These hypotheses are examined in the present study. DESIGN AND METHODS Using immunofluorometric assays, we studied 195 patients with primary breast cancer for the presence of p53 antibodies in serum and p53 protein accumulation in the corresponding tumor. Seventeen patients (9%) were p53 antibody-positive and 77 (40%) overexpressed p53. Ten of the 17 p53 antibody-positive patients had tumor p53 accumulation and 7 were negative for p53. Statistical analysis revealed a weak association between the presence of p53 antibodies and p53 protein accumulation (p = 0.05). Direct DNA sequencing of exons 1-11 of the p53 gene was performed for 16 p53 antibody-positive and 16 p53 antibody-negative patients. RESULTS Five of the seropositive and eight of the seronegative patients had a p53 gene mutation. Four of the five mutations in the p53 antibody-positive patients affected a Tyr residue, whereas none of the gene abnormalities in the seronegative patients had such an effect. CONCLUSIONS We conclude that p53 antibodies tend to develop in patients with tumor p53 accumulation, but p53 accumulation is neither sufficient nor necessary for the generation of the immune response. Further, p53 antibody-positive patients do not have higher frequency of p53 gene mutations than p53 antibody-negative patients, but the former patient group is associated with a Tyr substitution in the protein product.

Detection of the TP53 tumour suppressor gene product and p53 auto-antibodies in the ascites of women with ovarian cancer

Antibodies against the P53 tumour suppressor gene product are present in the serum of many cancer patients, but with varying frequencies ranging from 0 to 30%. Approximately 15-20% of patients with ovarian carcinoma develop auto-antibodies against p53 that circulate in the serum. Since many ovarian cancer patients develop ascites during their disease, we speculated that p53 antigen and/or p53 auto-antibodies may be present in this biological fluid. Ninety-six ascites fluids from women with primary ovarian cancer and one from a patient with cancer of the breast, which metastasised to the ovaries, were analysed for p53 auto-antibodies. Seventeen ascites fluids (18%) contained auto-antibodies. For 30 of these patients, serum was also available. Auto-antibodies were present in both serum and ascites in 6/30 patients; 22 patients were negative in both ascites and serum; and 2 patients had auto-antibodies only in their serum. All 97 ascites fluids were also analysed for p53 antigen and 7 (7%) were positive. None of the 17 p53 auto-antibodies-positive ascites fluids were positive for p53 antigen suggesting that p53 auto-antibodies may interfere with p53 antigen detection by capturing the antigen. In total, 24 patients (25%) had either p53 auto-antibodies or p53 protein in their ascites fluid. These data demonstrate that p53 auto-antibodies and/or p53 protein are present in ascites and may have some value for tumour diagnosis, prognosis or monitoring.

Identification of deletions and insertions in the p53 gene using multiplex PCR and high-resolution fragment analysis: application to breast and ovarian tumors.

We have developed a simple and highly efficient method to detect deletions and insertions in the p53 gene. All 11 exons of the p53 gene were amplified along with a control sequence in four multiplex PCR reactions in the presence of fluorescein‐labeled primers. The PCR products were resolved on an automated sequencing gel and the DNA fragments were detected by fluorescence. Using this method, we screened 7 DNA specimens from ovarian tumors, 19 from breast tumors, and 26 from normal breast tissues. No abnormality was found in any of the DNA samples extracted from the normal tissues. A 19 base pair deletion in exon 5 of the p53 gene was detected in one ovarian tumor. Insertions were identified in two breast and two ovarian tumors. The insertions were identical in 3 of these tumors and consisted of a 16 bp repeat within intron 3 of the p53 gene. It appears that the insertion within intron 3 may represent a hot spot for duplication of the normal sequence at that site. J. Clin. Lab. Anal. 12:250–256, 1998.

Characterization of the BRCA1-like immunoreactivity of human seminal plasma.

OBJECTIVES The subcellular localization of the breast cancer susceptibility gene product BRCA1 has been controversial. Discrepant results have been reported during the past 3 years, partially because of the unavailability of highly specific reagents for BRCA1 protein. Our objective was to characterize the BRCA1-like immunoreactivity that is detected in human seminal plasma by using monoclonal and polyclonal antibodies that are supposedly specific for BRCA1 protein. METHODS We used immunologic, chromatographic, and protein sequencing techniques to detect the immunoreactivity of BRCA1 in seminal plasma and to purify and partially identify the immunoreactive species. RESULTS We present data indicating that two BRCA1 antibodies, SG-11 and D-20, which were thought to be free of cross-reactivities, strongly interact with proteins present in human seminal plasma. This cross-reactivity is detectable even at seminal plasma dilutions as high as 10(6)-fold, and it is effectively blocked by peptides that capture the binding site of either SG-11 or D-20 antibodies. Purification and characterization of the immunoreactive compound revealed that this consists of a macromolecular complex that contains semenogelins. The D-20 polyclonal antibody was found to cross-react with purified semenogelins I and II; the SG-11 monoclonal antibody appeared to recognize a component of the macromolecular complex that was not semenogelin. CONCLUSIONS Our data demonstrate that the BRCA1 antibodies SG-11 and D-20 strongly interact with seminal plasma proteins and are not highly specific for BRCA1 protein. It is thus suggested that BRCA1 antibodies should be used with caution until reagents free of interference are developed and evaluated. In light of the very high cross-reactivity of the two antibodies with seminal plasma proteins, we recommend that new BRCA1 antibodies should be examined for cross-reactivity with seminal plasma proteins to verify specificity.

Fragment analysis of the p53 gene in ovarian tumors

he role of the p53 tumor suppressor gene inhuman carcinogenesis has been extensivelystudied. One central function of the p53 protein iscontrol of cellular growth after DNA damagethrough mechanisms involving growth arrest andapoptosis (1–3). These functions are believed to be atleast partially mediated by the ability of p53 to actas a transcription factor. Mutations in the p53 geneare found in most human malignancies and currentresearch is focusing on their role in cancer initiationand progression. p53 gene mutations can lead todefective cellular responses after DNA damage, dys-regulated cell growth, and tumor formation. Theidentification of p53 gene mutations in tumor cells isof diagnostic and therapeutic importance because:(a) tumors with a mutant p53 gene are usuallyresistant to certain chemotherapeutic agents or ra-diation; (b) a variety of tumors bearing a mutant p53gene have a less favorable prognosis than tumors ofthe same type with a wild-type p53 gene (4).The frequency of p53 gene mutations is high incancers of the colon (5), breast (6), lung (7), ovary (8),and brain (9). About 10% of the mutations aredeletions or insertions (10). Insertions range from 1to 14 nucleotides in length and in most cases, theinserted nucleotides duplicate the sequences of theneighboring region. Deletions range from 1 to 37nucleotides. Presence of deletions/insertions in 6 outof 11 newly established ovarian carcinoma cell lineshave been reported (11). In this report, we studiedthe presence of deletions and insertions in the p53gene in 89 primary ovarian tumors.

A Candidate New Gene on Human Chromosome 5q12 Contains a Motif That Is Found in Transcriptional Co-Activators

D our studies on prostate specific antigen (PSA) gene expression in lung carcinomas we have cloned and sequenced a 450 base pair (bp) novel sequence (1). Through genomic library screening and subcloning, we were able to extend the length of this sequence to 4.8 kilobases (kb). This entire sequence, which resides on chromosome 5q12, is now deposited in GenBank (accession number AF038385). In this article, we describe analysis of this genomic sequence with various computer programs and show that it contains an open reading frame encoding a proline and leucine-rich protein. The protein sequence contains a motif, LXXLL (L: leucine; X: any amino acid), that was found in the sequence of transcriptional co-activators (2). This is an important new family of nuclear proteins, which co-operate with steroid hormone receptors to regulate gene expression. The presence of this motif and the limited homology of the putative protein with another nuclear protein, which is a transcription factor, allow us to speculate that the newly cloned sequence is part of a gene encoding either a nuclear transcription factor or a transcriptional co-activator.