Chromosome 9p allelic loss and p16/CDKN2 in breast cancer and evidence of p16 inactivation in immortal breast epithelial cells.

To define the extent of involvement of chromosome 9p in breast carcinogenesis, we performed microsatellite length polymorphism analysis of markers spanning this region. Of 24 primary breast carcinomas analyzed, we observed a high frequency (58%) of loss of heterozygosity or allelic imbalance affecting subregion 9p21-22. Mutational analysis of CDKN2 (p16) was performed to determine whether this gene was the target of such alterations. Of 21 tumors analyzed, only 1 showed a mutation of probable consequence, suggesting that CDKN2 appears not to be the target of loss of heterozygosity and indicating the possible existence of another tumor suppressor gene within this region. Additionally, since it has been suggested that some CDKN2 deletions and mutations could be due to an in vitro phenomenon, four immortal breast cell lines derived from normal epithelium, MCF10F, MCF12F, 184A1, and 184B5, were examined for loss or mutation of CDKN2. Two lines (MCF10F and MCF12F) showed homozygous deletions of CDKN2, and one (184A1) revealed a hemizygous deletion and a nonsense mutation in the remaining allele. This could imply an important role of CDKN2 in the control of immortalization or in vitro adaptation and is the first evidence of such in nontumor-derived cell lines. Additionally, this is the first report of frequent loss of heterozygosity in the 9p21-22 chromosome subregion of uncultured primary breast tumors.

WWOX, the FRA16D gene, behaves as a suppressor of tumor growth.

We recently reported the cloning of WWOX, a gene that maps to the common fragile site FRA16D region in chromosome 16q23.3-24.1. It was observed that the genomic area spanned by WWOX is affected by chromosomal translocations and homozygous deletions. Furthermore, the high incidence of allelic loss in breast, ovarian, prostate, and other cancers affecting this region suggests that WWOX is a candidate tumor suppressor gene. Expression of WWOX is highly variable in breast cancer cell lines, with some cases showing low or undetectable levels of expression. In this report, we demonstrate that ectopic WWOX expression strongly inhibits anchorage-independent growth in soft agar of breast cancer cell lines MDA-MB-435 and T47D. Additionally, we observed that WWOX induces a dramatic inhibition of tumorigenicity of MDA-MB-435 breast cancer cells when tested in vivo. We also detected the common occurrence of aberrant WWOX transcripts with deletions of exons 5-8 or 6-8 in various carcinoma cell lines, multiple myeloma cell lines, and primary breast tumors. These aberrant mRNA forms were not detected in normal tissues. Interestingly, we further observed that proteins encoded by such aberrant transcripts display an abnormal nuclear localization in contrast to the wild-type WWOX protein that localizes to the Golgi system. Our data indicate that WWOX behaves as a potent suppressor of tumor growth and suggest that abnormalities affecting this gene at the genomic and transcriptional level may be of relevance in carcinogenesis.

Increased p16 expression with first senescence arrest in human mammary epithelial cells and extended growth capacity with p16 inactivation.

Aberrations affecting the tumor suppressor gene p16INK4a have been described for a variety of tumors. In breast cancer, approximately 50% of tumors show low or lack p16 expression. While evidence provided by some studies has implicated a possible role for p16 in normal replicative senescence, other studies have suggested that the Rb, pathway through which p16 functions, may not be involved in senescence control. Previously we observed that all immortal lines derived from normal mammary epithelium which were analysed for p16 displayed inactivation of this gene through distinct mechanisms, supporting p16 inactivation as a possible necessary event in escape from senescence. To further clarify this issue, we have analysed p16 expression in a panel of normal finite lifespan human mammary epithelial cells (HMEC) from initial propagation through growth arrest, using media which confer different replicative capacity. Approximately 10-25-fold increase in p16 expression was observed for all normal HMEC with initial onset of a senescence phenotype following 15-25 population doublings in culture. These cells also displayed expression of the senescence associated beta-galactosidase. Interestingly, HMEC with additional long term replicative capacity (approximately 80 population doublings) arose from these growth arrested cultures, showing lack of p16 expression. This extended growth capacity appears to be associated with a methylation phenomenon since treatment of these cells with the methylation inhibitor 5-aza-2-deoxycytidine resulted in growth arrest concurrent with reacquisition of p16 expression and senescence associated beta-galactosidase. Analysis of p21waf1 expression revealed no change in expression during growth in vitro. These results support p16INK4a as the 9p senescence gene and suggest a role for p16 loss in the escape from initial onset of senescence and in acquisition of an extended life span of human mammary epithelial cells.

Analysis of telomerase activity levels in breast cancer: positive detection at the in situ breast carcinoma stage.

Telomerase activity has been implicated to be associated with most human malignant tumors, including breast cancer. To evaluate possible associations with well-known prognostic factors in breast cancer, we performed a semi-quantitative analysis of telomerase activity levels using the very sensitive PCR-mediated telomeric repeat amplification protocol. Telomerase activity was detected in 99 of 104 breast cancer samples analyzed (95.2%), whereas no activity was detected in 10 of 10 adjacent nonmalignant breast tissues. Analysis of five breast fibroadenoma samples revealed telomerase activity in one (20%). In contrast to previous observations, we observed that 100% of stage I breast tumors were positive for telomerase activity. More interestingly, we detected telomerase activity in six of six ductal carcinoma in situ samples (i.e., stage 0). In our semiquantitative analysis of levels of enzymatic activity, we found no statistically significant correlation at the P < h 0.05 level between telomerase levels and lymph node metastasis status, estrogen and progesterone receptor status, tumor size, S-phase fraction, and ploidy. The only statistically significant correlation was found with patient age (rho = -0.3; P = 0.03). We observed no statistically significant difference in the telomerase activity levels of early tumors (stages 0 and 1) versus more advanced lesions (stages II to IV). Nevertheless, stage IV tumors displayed a tendency for higher telomerase activity levels. In summary, no clear association was observed between telomerase levels and known breast cancer prognostic indicators. However, telomerase detection by the telomeric repeat amplification protocol method, due to its high sensitivity, may be of value in early breast cancer diagnosis and detection, because our data indicate that telomerase reactivation appears to constitute a relatively early event in breast carcinogenesis.

Preferential loss of expression of p16(INK4a) rather than p19(ARF) in breast cancer.

The tumor suppressor p16(INK4a) has been shown to be inactivated in numerous cancer lines and primary tumors. Recently, we reported loss of heterozygosity of the region in which p16(INK4a) is located in more than one-half of primary breast tumors. However, mutational analysis of these same tumors revealed mutation of p16(INK4a) to be infrequent. Other possible modes of inactivation, such as de novo methylation and homozygous deletion, have since been shown to occur in numerous neoplasias. Furthering the complexity of this locus, a transcript overlapping the p16(INK4a) coding sequence and encoding a novel peptide with growth-suppressive activity, p19(ARF), has been described. To clearly elucidate the target of aberrations affecting this subchromosomal region and approximate frequency in breast cancer, we performed a comprehensive study including p16 deletion analysis by means of interphase chromosomal fluorescence in situ hybridization, methylation analysis of the first exon encoding p16(INK4a) (exon 1alpha), mutational analysis of exon 1beta by single-strand conformational polymorphism analysis of p19(ARF) transcripts, and expression of both alpha and beta transcripts by reverse transcription PCR. Homozygous deletion of p16, as determined by interphase chromosomal fluorescence in situ hybridization, was observed in 3 of 18 (17%) tumors analyzed, whereas de novo methylation of exon 1alpha was observed in an additional 17% (4 of 23). Reduced expression of p16(INK4a) was observed in 11 tumors (48%), including all those in which homozygous deletion or complete methylation was observed. No mutations of exon 1 beta were detected, and expression of its transcript was variable, with 13% demonstrating decreased expression and 17% demonstrating overexpression. These results further support p16(INK4a) as a target of inactivation in the 9p21 region for breast cancer.

A quantitative test for copper using bicinchoninic acid.

We describe a direct colorimetric assay for copper in serum and biological samples using 2,2'-bicinchoninic acid (BCA), a common reagent in most laboratories. BCA offers the advantage of being highly sensitive and specific for Cu(I) which rapidly forms an intense purple complex in the presence of BCA. The complex has peak absorbances at 562 and 354.5 nm with molar absorptivities of 7.7 x 10(3) liter mol-1 cm-1 and 4.6 x 10(4) liter mol-1 cm-1, respectively. Interference by other metal ions, pH, and detergents is minimal and the results correlate strongly with atomic absorption spectrophotometry.

The genetics of sporadic breast cancer.

Breast cancer is a complex disease in which numerous genetic aberrations occur. It is unclear which, if any, of these abnormalities are causative of breast tumorigenesis. However, on the basis of the currently accepted view of breast cancer as a multistep process, it is possible that specific abnormalities may be required in the progression from a normal breast epithelial cell to an invasive tumor cell. Figure 3 shows a schematic putative model of breast cancer progression based primarily on epidemiological and histopathological studies (Page and DuPont, 1992). Advances in methodology have allowed us to more precisely determine the approximate chronology of some of these aberrations and the possible roles each plays in the formation of malignancy. Simplistically, one could speculate that it is the early loss of cell cycle control in the presence of a mitogenic stimulus that allows a cell to divide unchecked. Such uncontrolled proliferation in the absence of wild type p53 would yield a high level of genomic instability. As proliferation continues, numerous additional chromosomal abnormalities occur, and increased tumor heterogeneity would be observed as distinct subpopulations emerge in the evolution toward a progressively more aggressive phenotype. However, much still remains to be learned to gain a full understanding of the key players behind the genetic evolution of breast cancer. Only by analyzing preinvasive and putative early stages of breast cancer will we be able to characterize the most probable sequence of genomic abnormalities.