Mara Hareuveni

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen: A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor‐associated antigen, H23, was compared to expression of genes coding for pS2, c‐erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non‐breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c‐erbB2, ER and pS2, respectively. Non‐malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.

A viral antigen as a marker for the prognosis of human breast cancer

An antigen present in human breast tumor cells, and which is immunologically related to the envelope protein (gp52) of murine mammary tumor virus, was used as a marker for the detection of breast cancer in an Israeli population. The results show that the antigen was detectable in 128 of 204 breast carcinomas tested (62.7%). The immunological reaction was not detected in normal breast tissue, benign breast tumors, ductal hyperplasia or in primary malignancies in other organs. A significantly higher percentage of cases with demonstrable antigen was found in Israeli women born in North Africa (78%) as compared to women of European origin (60.6%). The frequency of detection of the antigen was higher in stage IV (80%) as compared to stage I (15%), suggesting that the gp52 cross-reacting antigen is a marker for the severity of the disease. Moreover, a retrospective study of 97 cases of stage II breast cancer shows that if the antigen is detected at the time of mastectomy, one can usually predict an unfavorable prognosis. Survival data analysis indicates that patients without detectable antigen survived significantly longer than those with a detectable antigen.

H23 Monoclonal Antibody Recognizes a Breast Tumor Associated Antigen: Clinical and Molecular Studies

A monoclonal antibody (MoAb) H23 was generated in our laboratory against particles released by the T47D cell line. H23 MoAb recognized specific antigens in 90% of 590 breast tumor biopsies tested by the indirect immunoperoxidase test. Furthermore, the H23 MoAb detects antigens in sera and body fluids of patients with breast carcinoma. The level of serum antigen in 546 individuals tested correlates with the clinical status of the disease and with poor survival. The cDNA coding for the epitope recognized by H23 MoAb was isolated from a cDNA expression library and its sequence and orientation established. The nucleotide sequence showed that the cDNA insert was composed of 60 bp tandem repeats. We have analyzed the RNA isolated from primary human tumors, and it was demonstrated that breast carcinomas expressed the highest levels of mRNA species hybridizing with this cDNA. The gene coding for the cDNA was isolated from a genomic library and encompasses 7.5 kb. A 2.3 kb segment of this gene was found to be an array of tandem 60 bp repeat units whilst the remaining parts of the gene do not contain these repeats. The fact that these non-repeat sequences hybridize to identical mRNA species, has led to ongoing studies aimed at their further characterization.

Vaccination Against Breast Cancer — Studies in an Animal Model

One of the main mortality causes around the world are malignant diseases. In spite of the development of new cytotoxic drugs and new therapeutic protocols the death rate is still high due to failure of the response to treatment in metastatic cases1. Numerous factors set one tumor apart from another. Tumor specific markers are invaluable for diagnostic, prognostic and therapeutic purposes and enable the clinician to decide on treatment regimes.

Molecular Analysis of H23 Epithelial Tumor Antigen - Differentially Spliced Full Length cDNAs and Gene

The isolation and characterization of the complementary DNAs (cDNAs) and gene which code for an epithelial tumor antigen (H23-ETA), aberrantly expressed in human breast tumor tissue, are described here. A diversity of H23-ETA protein forms, is generated by a series of alternative splicing events that occur in regions located upstream and downstream to a central tandem 20 amino acid (aa) repeat array (TRA) that is rich in proline, serine and threonine residues. The upstream region shows that differential usage of alternative splice acceptor sites generates two protein forms containing putative signal peptides of varying hydrophobicities located at the NH2 terminus. The region downstream to the tandem repeat array indicates that one mRNA transcript is collinear with the gene and defines a 160 aa open reading frame (secreted or sec form). A second cDNA correlates with a mRNA that is generated by a series of splicing events and codes for 149 aa downstream to the TRA, identical with the aa sequence of the unspliced cDNA, after which it diverges and continues for an additional 179 aa. This sequence (transmembrane or tm form) contains a highly hydrophobic transmembrane domain of 28 aa followed by a hydrophilic “transfer-stop signal” (Arg Arg Lys) and a cytoplasmic domain of 72 aa. The various protein forms (alternative signal sequences, secreted and transmembrane) are likely routed to different cytoplasmic, cell membrane and extracellular compartments. Reverse PCR indicates that the relative ratios of the alternatively spliced forms vary in different epithelial tissues. To identify the individual protein species, monoclonal antibodies (mAb) are being generated against synthetic peptides unique to each form. The H23-ETA gene was also isolated and sequenced, demonstrating a putative promoter region that includes a ‘TATA’ box, Spl binding elements and an upstream putative hormone responsive element. Commensurate with these findings, H23-ETA expression was increased following hormonal treatment of BT549 breast tumor cells. These molecular studies have unravelled novel H23-ETA protein and gene structures, and facilitate future investigations that will focus on H23-ETA function and interaction with other cellular proteins.