Recognition and capture of breast cancer cells using an antibody-based platform in a microelectromechanical systems device.

Cancer is one of the most common diseases afflicting humans. The use of biomarkers specific for tumor cells has facilitated their identification. However, technology has not kept pace with the field of molecular biomarkers, leaving their potential unrealized. Here, we demonstrate the efficacy of recognizing and capturing cancer cells using an antibody-based, on-chip, microfluidic device. A cancer cell capture biochip consisting of microchannels of size 2.0 cm long and 500 microm wide and deep, was etched onto Polydimethylsiloxane. Epithelial membrane antigen (EMA) and Epithelial growth factor receptor (EGFR) were coated on the inner surface of the microchannels. The overall chip measured 2.0 cm x 1.5 cm x 0.5 cm. Normal and tumor breast cells in a phosphate buffered saline (PBS) suspension were flowed through the biochip channels at a rate of 15 microL/min. Breast cancer cells were preferentially captured and identified while most of normal cells passed through. The capture rates for tumor and normal cells were found to be >30% and <5%, respectively. This preliminary cancer cell capture biochip design supports our initial effort of moving a BioMEMS device, from the bench top to the clinic.

Microfluidic-based diagnostics for cervical cancer cells.

The use of biomarkers has facilitated the detection of specific tumor cells. However, the technology to apply these markers in a clinical setting has not kept pace with their increasing availability. In this project, we use an antibody-based microfluidics platform to recognize and capture cervical cancer cells. Because HPV-16 infection of cervical cells and up-regulation of alpha6-integrin cell surface receptors are correlated, we utilized alpha6-integrin as a capture antibody bound to the channel surface. Normal human glandular epithelial cells (HGEC), human cervical stromal cells (HCSC) and cervical cancer cells (HCCC) were suspended in PBS and flowed through the system. Greater than 30% of the cancer cells were captured while the capture of the normal cell types was less than 5%. The technique is sensitive and accurate. It is potentially useful in the detection of cervical cancer at all stages, as well as other of cancers with similar characteristics of cell surface antigen expression.

Alternative methods of extracting telomerase activity from human tumor samples.

Telomerase activity is present in approximately 85% of malignant cancers and >30% of premalignant lesions. Extraction of telomerase from solid tumors and tissues remains difficult and inconvenient due to the presence of PCR inhibitors. Here we show that PCR inhibitors are easily removed during an initial extraction, allowing detection of telomerase activity in subsequent extractions from the same sample. In addition, telomerase activity may be enriched and detected from very small samples in a large background by utilizing a biotin/strept-avidin coated, magnetic bead retrieval assay. Our results provide alternative methods for telomerase extraction from solid tumors and small samples that are more convenient and accurate.

Erwinia chrysanthemi strains cause death of human gastrointestinal cells in culture and express an intimin-like protein.

The bacterium Erwinia chrysanthemi is a model plant pathogen, responsible for causing cell death in plant tissue. Cell-wall depolymerizing enzymes and avirulence proteins essential for parasitism by this bacterium utilize dedicated type II and type III secretion systems, respectively. Although E. chrysanthemi is not recognized as a mammalian pathogen, we have observed that the bacterium can adhere to, cause an oxidative stress response in and kill cultured human adenocarcinoma cells. These bacteria express a surface protein that bears immunological identity to intimin, a protein required for full virulence of enterohemorrhagic and enteropathogenic Escherichia coli. A type III secretion mutant of E. chrysanthemi was observed to have a significantly lower capability of causing death than the wild-type strain in parallel cultures of human colon adenocarcinoma cells. These observations suggest that E. chrysanthemi has the potential to parasitize mammalian hosts as well as plants.

Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines.

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.

Characterization of paired tumor and non-tumor cell lines established from patients with breast cancer.

The goal of our study was to develop a panel of tumor cell lines along with paired non-malignant cell lines or strains collected from breast cancers, predominantly primary tumors. From a total of 189 breast tumor samples consisting of 177 primary tumors and 12 metastatic tissues, we established 21 human breast tumor cell lines that included 18 cell lines derived from primary tumors and 3 derived from metastatic lesions. Cell lines included those from patients with germline BRCA1 and FHIT gene mutations and others with possible genetic predisposition. For 19 tumor cell lines, we also established one or more corresponding non-malignant cell strains or B lymphoblastoid (BL) lines, which included 16 BL lines and 7 breast epithelial (2) or stromal (5) cell strains. The present report describes clinical, pathological and molecular information regarding the normal and tumor tissue sources along with relevant personal information and familial medical history. Analysis of the breast tumor cell lines indicated that most of the cell lines had the following features: they were derived from large tumors with or without axillary node metastases; were aneuploid and exhibited a moderate to poorly differentiated phenotype; were estrogen receptor (ER)- and progesterone receptor (PR)-negative; and overexpressed p53 and HER2/neu proteins. Of 13 patients with primary breast cancers receiving curative intent mastectomies, 7 were dead after a mean period of 10 months. Our panel of paired tumor and non-malignant cell lines should provide important new reagents for breast cancer research.

Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

Telomerase enzyme activity and RNA expression during the multistage pathogenesis of breast carcinoma.

Telomerase, an RNA-containing enzyme, is associated with cellular immortality and malignancy. We investigated the role of telomerase during the multistage pathogenesis of breast cancer. We used the semiquantitative, PCR-based telomeric repeat amplification protocol assay for enzyme activity (42 specimens from 42 patients) and a radioactive in situ assay for expression of its RNA component (human telomerase RNA; hTR) for the identification of telomerase-positive cells in archival resection samples (n = 67 from 39 patients). Low telomerase activity was detected in 1 (14%) of 7 samples of benign breast disease, in 4 (67%) of 6 fibroadenomas, in 11 (92%) of 12 carcinoma in situ (CIS) lesions, and in 16 (94%) of 17 invasive breast cancers. There was a progressive increase in the mean telomerase levels with progressive increase in severity of histopathological change (P < 0.05). Almost all of 67 resection samples expressed hTR, irrespective of histology. Expression was low to moderate in some samples of normal epithelium and nonproliferative fibrocystic changes. hTR expression was limited to epithelial cells; expression in stromal cells, including those in fibroadenomas, was negative. Increased hTR expression was observed in some foci of apocrine metaplasia and atypical hyperplasia. Increased hTR expression was also observed in all CIS and invasive lesions, although considerable heterogeneity was noted. Focal up-regulation was frequently noted in CIS lesions in the vicinity of invasive tumors. Thus, up-regulation of hTR may be a predictive marker for invasive tumor development.

Detection of telomerase activity in breast masses by fine-needle aspiration.

BACKGROUND: Telomerase is an RNA-dependent DNA polymerase that compensates for the telomere shortening that occurs in its absence. Reactivation of telomerase is thought to be an important step in cellular immortalization, and recent studies have indicated that telomerase activity is often detected in primary human malignancies. The clinical implications of telomerase activity in human tumors are currently under investigation. METHODS: Eighty-nine samples (46 FNAs and 43 gross tissue biopsies) from 44 patients with breast masses were analyzed prospectively for the presence of telomerase activity by a modification of the telomere repeat amplification protocol (TRAP). All samples were obtained directly from the excised mass at the time of specimen removal in the operating room. RESULTS: Telomerase activity was detected in 17 of 19 (90%) FNA samples and 15 of 18 (83%) invasive breast cancer tissue biopsies. Telomerase was also detected in 9 of 16 (56%) FNAs and 8 of 15 (53%) tissue biopsies from 16 fibroadenomas. Other benign proliferative lesions (n = 5) did not have detectable telomerase activity in either FNA or tissue specimens. FNA-TRAP results correlated with the gross tissue specimen TRAP results in 95% of all cases. CONCLUSION: The FNA-TRAP assay for telomerase detection is a highly sensitive and accurate method for the detection of telomerase activity in breast masses. Future application of these techniques should facilitate evaluation of telomerase as a tumor marker in the clinical management of breast and other solid malignancies.

Genomic instability and telomerase activity in human bronchial epithelial cells during immortalization by human papillomavirus-16 E6 and E7 genes.

Human papilloma virus types 16 and 18 contribute to the development of cervical carcinomas in which the E6 and E7 genes are frequently retained and expressed in the tumors. Our study explored the ability of the E6 and/or E7 genes to immortalize normal human bronchial epithelial (NHBE) cells and to reactivate telomerase expression in these cells. We have introduced the human papillomavirus type 16 E6 or E7 genes alone or in combination (E6/E7) into NHBE cells using the retroviral construct pLXSN. Cells expressing either the E6 or the E7 oncoproteins alone displayed an increased colony-forming efficiency and a slightly extended in vitro life span before entering a crisis, from which immortalized cell lines were not obtained. Telomerase activity was not detected in cells expressing either E6 or E7 individually. Cells expressing the E6/E7 oncoproteins in combination had a substantially increased life span before entering crisis. A subpopulation of these cells escaped from crisis and achieved 130 population doublings, suggesting immortalization. Telomerase activity was detected in these postcrisis cells, but was not detected prior to crisis. In addition, karyotypic analysis showed evidence of genomic instability in mass cultures as well as clones expressing E6, E7, or E6/E7. Abnormalities included numerous monosomies and trisomies, chromatid gaps and breaks, double minutes, and aberrant chromosomes. These results demonstrate that expression of E6 and/or E7 is sufficient to induce genomic instability and an extended life span to NHBE cells, but the presence of both E6 and E7, along with at least one additional genetic or epigenetic event achieved during crisis, was required for reactivation of telomerase and the immortalization in this human cell type.

Immortalization of human mammary epithelial cells transfected with mutant p53 (273his).

Normal human breast epithelial cells were transfected with expression vectors containing the p53 gene mutated at either codon 143, 175, 248 or 273, or by infection with a recombinant retroviral vector containing the p53 gene mutated at codons 143, 175, 248, or 273. The breast epithelial cells were monitored for extension of in vitro lifespan and immortalization. Expression of some, but not all, p53 mutants resulted in an extension of in vitro lifespan. Experiments with the p53 temperature sensitive mutant 143ala revealed that at 32 degrees C, the nonpermissive temperature, the growth of breast epithelial cells was inhibited. At 37 degrees C, the mutant conformation, there was increased proliferation of cells, resulting in extension of in vitro lifespan. Breast epithelial cells expressing p53 mutant 273his maintained DNA binding and transcriptional activities and one clone immortalized after a period of growth arrest (crisis). The progression of this immortalization event was characterized by the reactivation of telomerase using the telomeric repeat amplification protocol (TRAP), and terminal restriction fragment analysis (TRF). This is the first reported immortalization of human mammary epithelial cells transfected with a mutant 53.

Telomerase activity in human breast tumors.

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem." PURPOSE: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer. METHODS: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed. RESULTS: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02). CONCLUSION: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

p53 levels in human mammary epithelial cells expressing wild-type and mutant human papillomavirus type 16 (HPV-16) E6 proteins.

Human fibroblast cells must overcome both the M1 and the M2 stages of cellular senescence to immortalize, at which point cells almost always express telomerase activity. The human papillomavirus (HPV) oncoproteins, HPV-16 E6 and E7, can block the progression to senescence in fibroblasts by associations with p53 and pRb, respectively. Human mammary epithelial (HME) cells require only HPV-16 E6 to bypass M1, suggesting that pRb may not have a direct role in HME cells senescence. In the present report, we show that only wild-type HPV-16 E6 allows complete degradation of p53, immortalization and reactivation of telomerase activity in HME cells. These results suggest that the ability of HPV-16 wild-type and mutant E6 proteins to degrade p53 in intact HME cells and keratinocytes does not completely correlate with their ability to degrade p53 in a cell-free system. This discrepancy between in vitro and in vivo p53 degradation may be biologically significant and may provide insight into the susceptibility of certain human cells and tissues for reactivation of telomerase and immortalization.

Spontaneous in vitro immortalization of breast epithelial cells from a patient with Li-Fraumeni syndrome.

Individuals with germ line mutations in the p53 gene, such as Li-Fraumeni syndrome (LFS), have an increased occurrence of many types of cancer, including an unusually high incidence of breast cancer. This report documents that normal breast epithelial cells obtained from a patient with LFS (with a mutation at codon 133 of the p53 gene) spontaneously immortalized in cell culture while the breast stromal fibroblasts from this same patient did not. Spontaneous immortalization of human cells in vitro is an extremely rare event. This is the first documented case of the spontaneous immortalization of breast epithelial cells from a patient with LFS in culture. LFS patient breast stromal fibroblasts infected with a retroviral vector containing human papillomavirus type 16 E7 alone were able to immortalize, whereas stromal cells obtained from patients with wild-type p53, similarly infected with human papillomavirus type 16 E7, did not. The present results indicate a protective role of normal pRb-like functions in breast stromal fibroblasts but not in breast epithelial cells and reinforces an important role of wild-type p53 in the regulation of the normal growth and development of breast epithelial tissue.

Loss of heterozygosity at chromosome 1q loci in rat mammary tumors.

To better characterize abnormalities affecting rat chromosome 1 during mammary carcinogenesis, tumors were induced by nitrosomethylurea in F1 hybrid rats polymorphic at multiple chromosome 1 loci. By means of restriction fragment length polymorphism and microsatellite length polymorphism analyses, we observed loss of heterozygosity or allelic imbalance affecting various loci on the q arm of chromosome 1 in a high percentage of the 49 tumors analyzed. Fifty percent of the tumors showed loss or imbalance affecting the most distal (1q55) INS1 (rat insulin 1 gene) locus. The MT1PA (metallothionein-1 pseudogene a) locus was observed to be affected in 58% of tumors induced in BUF/NCr x ACI/Vsp rats. Most of the losses appeared to have occurred by mitotic recombination. No parental bias was observed on the affected chromosome 1. Tumors were also screened for mutations in codon 12 of the Ha-ras-1 gene, which is located on 1q. We observed an association between the presence of mutation and allelic imbalance. These studies confirm our previous cytogenetic observations of a high level of nonrandom instability affecting rat chromosome 1 during mammary carcinogenesis. The observed loss of heterozygosity may indicate the existence of a putative tumor suppressor gene within the distal half of the 1q arm. These abnormalities, however, could also be related to the early stages of Ha-ras amplification.

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and their receptors in human breast cells and tissues: alternative receptors.

We sought to determine whether the hepatocyte growth factor/scatter factor (HGF/SF)- and keratinocyte growth factor-receptor systems were expressed in normal breast cells, breast carcinoma cell lines, normal breast tissues, and breast cancer tissues. Reverse transcriptase-polymerase chain reaction and hot blotting were used to detect HGF, HGF/SF (met) receptor, KGF, and KGF receptor mRNAs in human mammary epithelial (HME) and stromal (HMS) cells. We also examined breast carcinoma (MDA-MB-157, SCC 38, and SCC 70) and spontaneously immortalized breast epithelial (HMT 3522) cell lines, as well as normal breast and breast carcinoma tissues. PCR products were also confirmed by nucleic acid sequencing. The effects of HGF and KGF, compared to EGF and heparin-binding EGF, on the proliferation of normal human mammary epithelial cells in serum-free defined medium was determined by cell counting. HGF and KGF mRNAs were detected in HMS cells, but not HME cells. KGF receptor mRNA was detected in HME cells, but not HMS cells. HGF/SF receptor mRNA was detected in both HME and HMS cells. mRNAs were also detected in normal breast and breast carcinoma tissues, as well as breast carcinoma and transformed breast epithelial cell lines. Alternative cDNA sequences that are predicted to code for a soluble KGF receptor and a membrane bound, truncated HGF/SF receptor were detected in breast epithelial cells and breast tissues. HGF and KGF maintained viability and stimulated proliferation of HME cells.

Systematic HRAS amplification in ovary-independent mammary tumors: correlation with progressively anaplastic phenotypes.

Progression of well differentiated rat mammary adenocarcinomas to very anaplastic phenotypes was found to correlate with a systematic and significant amplification of a mutant HRAS allele. Tumors with high amplification levels of this oncogene were analyzed by chromosomal in situ hybridization; in four of the cases the amplified sequences did not reside at the native chromosome 1 locus but were localized in a novel marker chromosome. The model described has potential as a reproducible system for the study of the chromosomal and cellular mechanisms operative "in vivo" for oncogene amplification.

Nonrandom abnormalities involving chromosome 1 and Harvey-ras-1 alleles in rat mammary tumor progression.

Little is known about the role of chromosomal abnormalities in the widely used models of rat mammary carcinogenesis. In this study, we cytogenetically analyzed nitrosomethylurea-induced rat mammary adenocarcinomas at different time points of development. As tools to study more advanced stages of malignant progression, we also analyzed the cytogenetic progression of tumors transplanted into younger syngeneic hosts, and of tumors that did not regress or that developed after host ovariectomy. Our results indicate that rat mammary adenocarcinomas appear to start development as diploid lesions with cytogenetically "normal" karyotypes. However, upon progression, tumors showed coexistence of normal diploid clones with abnormal clones bearing specific abnormalities affecting mainly chromosomes 1 and 15. Almost every ovary-independent tumor presented stem lines with specific nonrandom chromosomal abnormalities. Numerical chromosomal abnormalities such as specific trisomies started to develop mainly after subsequent in vivo transplantations. The abnormalities affecting chromosome 1 observed in many tumors were: (a) interstitial deletions and breakpoints for translocation in region 1q22; and (b) partial or complete overrepresentation of chromosome 1 in the form of direct duplication of region 1q22q43 or as trisomy 1. Interestingly, Harvey-ras-1 gene maps to rat chromosome 1, and by Southern analysis we observed that 4 of 8 primary tumors and 6 of 9 ovary-independent tumors showed considerable loss of Harvey-ras-1 signal indicating probable allele loss. However, analyses of some tumor transplants in more advanced stages of progression showed, paradoxically, an increased copy number of the Harvey-ras-1 oncogene coinciding with the presence of the direct duplication observed in chromosome 1 or with trisomy 1 as possible mechanisms for gene amplification. Interestingly, rat chromosome 1 is the homologue to human chromosome 11, and in numerous cases of human breast cancer loss of heterozygosity of several genes in chromosome 11p15.5 has been reported. Some of the rat chromosome 1 abnormalities observed may be equivalent to those affecting 11p15 in human tumors. We also observed 8 tumors with abnormalities affecting chromosome 15. At least 3 genes of interest in breast cancer have been previously mapped to that rat chromosome. The similarities observed with human breast cancer may point to common mechanisms of tumor progression in both species.

Chromosomal localization of the rat Harvey-ras-1 gene (HRAS) by in situ hybridization.

The rat Harvey-ras-1 protooncogene (HRAS) has previously been assigned to rat chromosome 1. In this study we further refine its localization to region 1q41-->q42 through the use of fluorescent in situ hybridization.