Part I. Molecular and cellular characterization of high nitric oxide-adapted human breast adenocarcinoma cell lines.

There is a lack of understanding of the casual mechanisms behind the observation that some breast adenocarcinomas have identical morphology and comparatively different cellular growth behavior. This is exemplified by a differential response to radiation, chemotherapy, and other biological intervention therapies. Elevated concentrations of the free radical nitric oxide (NO), coupled with the up-regulated enzyme nitric oxide synthase (NOS) which produces NO, are activities which impact tumor growth. Previously, we adapted four human breast cancer cell lines: BT-20, Hs578T, T-47D, and MCF-7 to elevated concentrations of nitric oxide (or high NO [HNO]). This was accomplished by exposing the cell lines to increasing levels of an NO donor over time. Significantly, the HNO cell lines grew faster than did each respective ("PARENT") cell line even in the absence of NO donor-supplemented media. This was evident despite each "parent" being morphologically equivalent to the HNO adapted cell line. Herein, we characterize the HNO cells and their biological attributes against those of the parent cells. Pairs of HNO/parent cell lines were then analyzed using a number of key cellular activity criteria including: cell cycle distribution, DNA ploidy, response to DNA damage, UV radiation response, X-ray radiation response, and the expression of significant cellular enzymes. Other key enzyme activities studied were NOS, p53, and glutathione S-transferase-pi (GST-pi) expression. HNO cells were typified by a far more aggressive pattern of growth and resistance to various treatments than the corresponding parent cells. This was evidenced by a higher S-phase percentage, variable radioresistance, and up-regulated GST-pi and p53. Taken collectively, this data provides evidence that cancer cells subjected to HNO concentrations become resistant to free radicals such as NO via up-regulated cellular defense mechanisms, including p53 and GST-pi. The adaptation to NO may explain how tumor cells acquire a more aggressive tumor phenotype.

Part II. Mitochondrial mutational status of high nitric oxide adapted cell line BT-20 (BT-20-HNO) as it relates to human primary breast tumors.

Mitochondria combine hydrogen and oxygen to produce heat and adenosine triphosphate (ATP). As a toxic by-product of oxidative phosphorylation (OXPHOS), mitochondria generate reactive oxygen species (ROS). These free radicals may cause damage to mitochondrial DNA (mtDNA) and other molecules in the cell. Nitric oxide (NO) plays an important role in the biology of human cancers, including breast cancer; however, it is still unclear how NO might affect the mitochondrial genome. The aim of the current study is to determine the role of mtDNA in the breast oncogenic process. Using DNA sequencing, we studied one breast cancer cell line as a model system to investigate the effects of oxidative stress. The BT-20 cell line was fully adapted to increasing concentrations of the NO donor DETA-NONOate and is referred to as BT-20-HNO, a high NO (HNO) cell line. The HNO cell line is biologically different from the "parent" cell line from which it originated. Moreover, we investigated 71 breast cancer biopsies and the corresponding noncancerous breast tissues. The free radical NO was able to generate somatic mtDNA mutations in the BT-20-HNO cell line that were missing in the BT-20 parent cell line. We identified two somatic mutations, A4767G and G13481A, which changed the amino acid residues. Another two point mutations were identified in the mtDNA initiation replication site at nucleotide 57 and at the 'hot spot' cytidine-rich D300-310 segment. Furthermore, the NO regulated the mtDNA copy number and selected different mtDNA populations by clonal expansion. Interestingly, we identified eight somatic mutations in the coding regions of mtDNAs of eight breast cancer patients (8/71, 11.2 %). All of these somatic mutations changed amino acid residues in the highly conserved regions of mtDNA which potentially leads to mitochondrial dysfunctions. The other two somatic mtDNA mutations in the displacement loop (D-loop) region [303:315 C(7-8)TC(6) and nucleotide 57] were distributed among 14 patients (14/71, 19.7 %). Importantly, of these 14 patients, six had mutations in the p53 gene. These results validate the BT-20 parent/HNO cell line model system as a means to study ROS damage in mtDNA, as it parallels the results found in a subset of the patient population.

Part III. Molecular changes induced by high nitric oxide adaptation in human breast cancer cell line BT-20 (BT-20-HNO): a switch from aerobic to anaerobic metabolism.

Nutrient deprivation and reactive oxygen species (ROS) play an important role in breast cancer mitochondrial adaptation. Adaptations to these conditions allow cells to survive in the stressful microenvironment of the tumor bed. This study is directed at defining the consequences of High Nitric Oxide (HNO) exposure to mitochondria in human breast cancer cells. The breast cancer cell line BT-20 (parent) was adapted to HNO as previously reported, resulting in the BT-20-HNO cell line. Both cell lines were analyzed by a variety of methods including MTT, LDH leakage assay, DNA sequencing, and Western blot analysis. The LDH assay and the gene chip data showed that BT-20-HNO was more prone to use the glycolytic pathway than the parent cell line. The BT-20-HNO cells were also more resistant to the apoptotic inducing agent salinomycin, which suggests that p53 may be mutated in these cells. Polymerase chain reaction (PCR) followed by DNA sequencing of the p53 gene showed that it was, in fact, mutated at the DNA-binding site (L194F). Western blot analysis showed that p53 was significantly upregulated in these cells. These results suggest that free radicals, such as nitric oxide (NO), pressure human breast tumor cells to acquire an aggressive phenotype and resistance to apoptosis. These data collectively provide a mechanism by which the dysregulation of ROS in the mitochondria of breast cancer cells can result in DNA damage.

Expression of the adenocarcinoma-related antigen recognized by monoclonal antibody 44-3A6 in salivary gland neoplasias.

The monoclonal antibody 44-3A6 detects a cell-surface protein that has been shown to be a useful marker in distinguishing adenocarcinomas from other histologic tumor types in a variety of tissues. The objective of this study was to determine whether 44-3A6 could be used as a tool in the classification of salivary gland neoplasms. These complex tumors share overlapping pathologic features but distinct clinical outcomes. This study used 44-3A6 to immunohistochemically describe the pattern and frequency of this antigen in salivary gland neoplasms. Formalin-fixed, paraffin-embedded tissue sections of 22 benign and 26 malignant salivary tumors were evaluated. The patient population consisted of 25 (52.1%) women and 23 (47.9%) men selected from archival pathology files to reflect a range of salivary gland diseases. Normal surrounding salivary glands were found to have intense focal staining almost exclusively localized to ductal luminal cells. There was little staining of either myoepithelial or acinar cells. A wide spectrum of expression was found between and within tumor types, but a trend toward more expression of this antigen with decreasing differentiation was seen. A significant increase in staining was also seen in those tumors with ductal differentiation (n = 41) as opposed to those with predominantly acinar (i.e., acinic cell carcinoma) or myoepithelial (i.e., myoepithelioma; n = 8) differentiation (2.6 vs. 1.3, p < 0.05). No correlation was found between staining intensity and facial paralysis, pain, skin involvement, TNM stage, residual disease, or disease-free or total survival. Therefore this antigen appears to designate a duct luminal phenotype in normal and neoplastic salivary tissues.

Reduced in vivo local recurrence with contact neodymium: Yttrium-Aluminium-Garnet (Nd:YAG) laser scalpels.

BACKGROUND AND OBJECTIVE: Local recurrence after surgical resection for breast cancer is a significant problem and is often not controlled by radiation or chemotherapy treatments. Local recurrence is thought to be, at least in part, due to residual disease, and/or due to the contamination of the surgical field during resection. STUDY DESIGN MATERIALS AND METHODS: To address this later concern, we defined a model system using the mouse mammary cell line, EMT6. Using this model system, we have directly compared the rate of local recurrence of two different surgical approaches. One approach employed the use of traditional surgical instruments, and the other used a comparatively new contact Nd:YAG laser system. Tumor-bearing animals (242) were randomized into three groups. One group consisted of 50 animals that were not treated; 103 animals were randomized into a treatment group that received surgical resection using traditional instruments; 89 animals were resected using the contact laser system. In both surgical procedures, an intentional incision was made through the tumor and then through an uninvolved portion of the surgical field in an attempt to "seed" the incision using the contaminated surgical instrument. RESULTS: Twenty-one of the 103 scalpel-treated animals had local recurrence; only seven of the 89 laser-treated animal had local recurrence. The untreated group died of disease within 8 weeks. In the treatment groups, recurrences were palpable within 1 week. At the time of death for all groups, no metastatic lesions were noted. CONCLUSION: These findings support the conclusions that the EMT6 cell line is a useful model to study local recurrence and that contact laser surgery provides about a 50% improvement in the control of local disease in vivo (P < 0.05).

Adaptation of the diphenylamine (DPA) assay to a 96-well plate tissue culture format and comparison with the MTT assay.

A simple spectrophotometric method for measuring DNA in proliferating cells is described. The method is an adaptation of the widely used diphenylamine (DPA) reaction to examine DNA in cells grown in a 96-well plate. This assay was capable of detecting as little as 10 ng DNA and could be used to measure DNA in stored as well as viable tissue samples. The DPA assay paralleled the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay of mitochondrial reductase in A549 cells, a human lung cancer cell line; in EMT6 cells, a mouse breast cancer cell line; and in a primary cell culture of neonatal rat astrocytes. Over several days of proliferative growth in tissue culture, the ratio of MTT to DPA remained constant. Since the DPA assay and MTT assay measured different parameters of the same cells, they could be employed as complementary spectrophotometric assessments of cell growth on a 96-well plates using the same automated enzyme-linked immunosorbent assay plate reader.

A new method for the detection of viable cells in tissue sections using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT): an application in the assessment of tissue damage by surgical instruments.

This report describes a new method using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to visualize live viable tissue at the microscopic level. The visualization of the MTT stained tissue provides a metabolic assessment of tissue damage, and can be utilized as an extension of conventional hematoxylin-eosin (H & E) staining. In this report, several tissues were studied with MTT and H & E staining after incisions had been made by a variety of surgical dissecting instruments. A marked improvement in the detection of tissue damage was seen using MTT, regardless of how the damage was caused, i.e., physical, heat, or photon energy. In addition, a distinct zone of damage not noted on conventionally prepared and stained tissues is readily apparent. Thus MTT staining will have utility in both clinical and research studies, concerned with assessing the viability of tissues.

Reduced tumor cell transfer with contact neodymium-yttrium-aluminium garnett laser scalpels.

The local recurrence of tumor growth after surgery is thought, in part, to be a consequence of seeding of tumor cells from the primary lesion. This study was directed at determining if the contact Nd:YAG laser provides any advantage over the use of traditional scalpel dissection for tumor resection. Five human tumor tissue culture cell lines were studied in this report. They included MCF-7 (breast), HeLa (cervical), SW-780 (bladder), HT29 (colon), and A549 (lung). Using a scalpel blade or contact laser scalpel (with or without laser energy), the ability to transfer viable cells from dense cell stocks to new tissue culture wells was tested. Using the A549 cell line, the extent that these instruments were able to "seed" tumor cells was also assessed in a soft agar, in vitro, "incision" model. Results from these studies suggest that the contact laser scalpel has a significantly lower potential to transmit tumor cells, when compared to traditional scalpel blades.