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.

The effect of proton pump inhibitors on the human microbiota.

Proton pump inhibitors (PPIs) are commonly used to treat acid-related diseases, most notably gastroesophageal reflux disease. PPIs are designed to shut down the gastric proton pump (H+/K+-ATPase) of parietal cells, thereby raising the pH of the stomach. While effective, a number of side effects have been associated with PPI use. Naturally occurring bacteria, some of which are acid-producing and contain ATPase enzymes, have also been found within the stomach, upper gastrointestinal tract, and oral cavity. Likewise, a number of fungi are known to inhabit the human body; some of these fungi contain H+-ATPase enzymes. Recent literature has suggested that PPIs may be inadvertently affecting these bacteria and fungi in two different ways: 1) PPIs may directly target the proton pumps of the bacteria and fungi, and/or 2) PPIs may indirectly affect the microenvironment of the flora via changes in pH. These unintended interactions are exasperated by the systemic distribution of PPIs throughout the body and may potentially lead to some of the side effects observed with PPI use. Herein we summarize what is currently known about the interactions between the PPIs and the natural human microbiota.

Expression of nitric oxide synthase type 3 in reflux-induced esophageal lesions.

BACKGROUND: The expression of endothelial constitutive nitric oxide synthase (NOS3) by squamous dysplasia and carcinomas of the head and neck has previously been described. We sought to compare NOS3 expression in squamous mucosa, glandular metaplasia, and adenocarcinoma of the esophagus. METHODS: Forty paraffin-embedded specimens from 20 patients with adenocarcinoma were stained with anti-NOS3 monoclonal antibody. The percentage of cells stained and the intensity of staining were determined for squamous epithelium, metaplasia, and adenocarcinoma. Staining characteristics were statistically analyzed according to clinical variables. RESULTS: NOS3 expression was significantly higher in adenocarcinoma and squamous epithelium compared with glandular metaplasia. Among the carcinomas, larger tumor size (T3/4), nodal positivity, and advanced TNM stage (III/IV) significantly correlated with increased NOS3 expression. CONCLUSIONS: NOS3 is expressed in reflux-induced lesions of the esophagus. Glandular metaplasia shows basal levels of NOS3 that significantly increase with malignant transformation and tumor progression. The role of free radicals in carcinogenesis is being actively studied.

Glutathione S-transferase pi in squamous cell carcinoma of the head and neck.

OBJECTIVES/HYPOTHESIS: Oxidative/reductive (redox) DNA damage from radical species such as nitric oxide (NO*) are increasingly being implicated in the development of cancer. Moreover, redox-protective cellular mechanisms, such as glutathione S-transferase, may determine cellular susceptibility to this redox-mediated damage. METHODS: Formalin-fixed, paraffin-embedded tissue samples of 11 normal oral mucosa, 15 reactive/dysplastic lesions, and 131 head and neck squamous cell carcinomas (HNSCCs) were immunohistochemically stained using a polyclonal antibody against glutathione S-transferase pi (GST-pi). Slides were reviewed in a blinded fashion by the study pathologist (G.K.H.) and intensity was graded, noting the pattern of immunostaining. These staining characteristics were compared with those obtained using monoclonal antibodies against endothelial constitutive nitric oxide synthase (ecNOS) and nitrotyrosine, a marker of NO*'s pathological nitrosylation of proteins on serial sections of the same tissue. Patient charts were reviewed and clinical data collected. RESULTS: The expression of GST-pi was significantly increased in reactive/dysplastic and HNSCC samples compared with normal squamous mucosa (P < .001 for both). Furthermore, among the carcinomas the expression of GST-pi was significantly increased in higher-grade lesions (P < .02). The expression of GST-pi correlated highly with the expression of ecNOS and nitrotyrosine (P < .0001 for both). CONCLUSIONS: These findings demonstrate that GST-pi is upregulated in conjunction with the NO*-generating ecNOS isoform and implicate GST-pi in protecting HNSCC from the cytotoxic effects of high concentrations of NO* found in the tumor bed.

Immunohistochemical localization of modified C-reactive protein antigen in normal vascular tissue.

BACKGROUND: The prototypic acute phase reactant, C-reactive protein (CRP), is a serum soluble, cyclic pentameric protein, the concentration of which increases markedly within hours of any tissue-damaging, inflammatory event. However, upon dissociation of its pentameric quaternary structure, CRP subunits undergo a spontaneous and irreversible conformational change. The resulting molecule, termed modified CRP or mCRP, has reduced aqueous solubility and a propensity to aggregate into a matrix-like lattice structure. METHODS: Using monoclonal antibodies, normal human tissues were immunohistochemically screened for the presence of CRP as well as mCRP antigens. RESULTS: Significant levels of mCRP were detected in the walls of blood vessels associated with normal human tissues. These data indicate that mCRP is a naturally occurring form of CRP and that it is a tissue-based rather than serum-based molecule. SIGNIFICANCE: This report describes the localization of a stable form of CRP, mCRP, in blood vessels associated with normal human tissues.

Increased protein nitrosylation in head and neck squamous cell carcinogenesis.

BACKGROUND: Nitric oxide (NO.) and its metabolic byproducts are implicated in carcinogenesis. We examined a marker of NO.-species' pathologic protein nitrosylation, nitrotyrosine, during head and neck squamous cell carcinoma (HNSCCa) development. Materials and Methods Paraffin-embedded tissue samples of normal oral mucosa, squamous hyperplasia/dysplasia, and HNSCCa were immunohistochemically analyzed for staining intensity using an antinitrotyrosine monoclonal antibody, and correlated with retrospective clinical data. RESULTS: Significantly higher staining was noted in reactive, dysplastic and HNSCCa samples compared with samples of normal mucosa. Additionally, significant differences in staining were found between various primary sites of the upper aerodigestive tract. Lastly, individual inflammatory cells also stained intensely. CONCLUSIONS: Increasing amounts of nitrotyrosine staining were found in reactive/dysplastic and HNSCCa lesions compared to normal squamous mucosa. Inflammatory cell staining implicates NO. as a possible mediator of immunosuppression. Given these findings, the role of NO. in mutations leading to and the immunosuppression found in HNSCCa warrants further investigation.

The yin and yang of nitric oxide: reflections on the physiology and pathophysiology of NO.

Nitric oxide (NO.) is an arginine-derived nitrogen-based radical that is rapidly becoming one of the most important molecular species to be discovered. Over the past decade, an explosion of evidence has revealed the extreme complexity of function of this seemingly simple inorganic molecule. It is now evident that NO. demonstrates a functional dualism, playing a pivotal role in numerous physiologic and pathophysiologic processes. Whether this molecule is beneficial or detrimental is dependent upon the tissue of generation, the level of production, the oxidative/reductive (redox) environment in which this radical is generated, and the presence or absence of NO. transduction elements. Nitric oxide is generated by three independent isoenzymes that resemble the p-450 enzyme superfamily in both form and function. It ultimately alters enzymatic function through covalent modification, redox interactions, and interactions with metallic functional centers. This radical is a key figure in a number of pathophysiologic processes by means of similar yet uncoordinated interactions. In consideration of the already broad spectrum of roles attributed to NO., it seems highly likely that this molecule will be implicated in an ever widening variety of functions relative to the practice of otolaryngology-head and neck surgery. This article reviews the enzymology, signal transduction mechanisms, physiology, and pathophysiology of NO. as it pertains to head and neck cancer.

Nitric oxide synthase type 3 is increased in squamous hyperplasia, dysplasia, and squamous cell carcinoma of the head and neck.

The implication of nitric oxide (NO*) in the multistep process of carcinogenesis prompted us to examine the expression of endothelial constitutive nitric oxide synthase (NOS3) in head and neck squamous cell carcinoma (HNSCCa). Eleven paraffin-embedded samples of normal oral mucosa, 3 reactive oral lesions, 13 samples of squamous dysplasia, and 120 specimens of HNSCCa were immunostained with an anti-NOS3 monoclonal antibody and graded on a 0 to 4+ scale of intensity. Normal squamous mucosa demonstrated very little NOS3 expression. Areas of normal mucosa, reactive mucosa, and dysplastic lesions associated with inflammation tended to demonstrate regional expression of NOS3. Reactive mucosal lesions, squamous dysplasia, and HNSCCa demonstrated a significant (p<.0001) increase in global expression of NOS3. Therefore, NOS3 is expressed very little in histologically normal squamous mucosa, while squamous hyperplasia, dysplasia, and HNSCCa express significantly more NOS3. Regional variation in NOS3 expression appears to be associated with perilesional inflammation.

Inhibition of mouse mammary adenocarcinoma (EMT6) growth and metastases in mice by a modified form of C-reactive protein.

Mice were injected in the hind limb with a mouse mammary adenocarcinoma cell line, EMT6, and tumor growth at the primary site as well as the incidence of lung metastases were measured. Groups of animals were treated with the acute-phase reactant C-reactive protein, (native-CRP), or a conformationally modified form of CRP (mCRP) made by dissociating CRP subunits under chelating, denaturing conditions. Each form of CRP was injected (intravenously) through the tail vein, encapsulated in large unilamellar lipid vesicles made by an extrusion technique (LUVETs). mCRP was also injected without the LUVET carrier. Mice not treated, or treated with LUVETs alone, exhibited both progressive tumor growth at the primary site and a high incidence of metastatic lung tumors quantified at necropsy. Treatment with native-CRP encapsulated in LUVETs had little or no effect on either tumor growth or metastases. Treatment with mCRP, however, alone or encapsulated in LUVETs, effectively slowed or stopped the progression of tumor growth, and in some mice, showed a decrease in tumor size. After cessation of mCRP injections, tumor growth resumed at a rate comparable to that measured in untreated animals. Fifty to 85% of mice treated with mCRP or mCRP in LUVETs developed necrotic lesions at the primary tumor site within 24-48 h following the initial injection of protein. Furthermore, at necropsy, only 6% of mice treated with mCRP in LUVETs and 40% of mice treated with mCRP alone showed evidence of lung metastases compared to 67-80% of animals in no-treatment, native-CRP in LUVETs and in LUVET control group animals. These results show that the prototypic acute-phase reactant, CRP, has therapeutic anticancer and antimetastatic activity only when the native pentameric subunit structure is dissociated to form the mCRP conformer.

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.

Endothelial constitutive nitric oxide synthase (ecNOS) localization in normal and neoplastic salivary tissue.

BACKGROUND: Nitric oxide (NO.) has been implicated in the process of carcinogenesis in various organs. This study was designed to investigate the expression of endothelial constitutive nitric oxide synthase (ecNOS) in normal and neoplastic salivary tissues. METHODS: Paraffin-embedded tissue from 48 salivary tumors and adjacent non-neoplastic tissue was immunohistochemically evaluated for both frequency (percentage) and intensity (1-4+) of staining using a commercially available anti-ecNOS monoclonal antibody. RESULTS: Expression of ecNOS was predominantly localized to vascular endothelium, skeletal muscle, and to salivary duct luminal epithelium in normal salivary tissue (n = 37). All salivary tumors demonstrated at least 1 + cytoplasmic staining for ecNOS without apparent correlation to most clinical parameters. A tendency toward increased frequency and intensity of ecNOS expression in oncocytic cells, relative to cells with myoepithelial or acinar differentiation, was noted. CONCLUSIONS: Expression of ecNOS is localized to the luminal cells of normal salivary ducts. Limited expression of ecNOS was found in all the salivary gland tumors examined. This suggests a common histogenesis for this diverse group of tumors, which may reflect different degrees of differentiation toward luminal duct epithelium. The possible role of ecNOS and NO. in salivary gland carcinogenesis is intriguing and warrants further study.

Scaphocephaly: premature closure of the sagittal suture: a localized disorder of cellular metabolism?

Osteoblasts derived from sagittal sutures with premature synostosis, noninvolved coronal sutures, and normal frontal bone were harvested and cultured as cells in an attempt to determine if osteoblasts at the site of premature fusion exhibited altered in vitro cellular dynamics. Basal metabolic parameters of cellular growth and the production of metabolites, including osteocalcin, alkaline phosphatase, platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta), PDGF-beta receptors, epidermal growth factor (EGF) receptors, and fibroblast growth factor (FGF) were characterized. Osteoblasts harvested from sagittal sutures (sagittal osteoblasts) exhibited altered cellular growth and indices of cellular metabolism when compared with osteoblasts derived from patent coronal sutures (coronal osteoblasts) and frontal bone (frontal osteoblasts). Sagittal osteoblasts grew at a significantly increased rate and produced significantly more osteocalcin and less alkaline phosphatase than the coronal and frontal osteoblasts (p < 0.05). Significant variations in the production of EGF receptors were also noted between the sagittal osteoblasts and the coronal and frontal osteoblasts (p < 0.05). The osteoblasts from coronal sutures exhibited similarities in cellular growth and cellular metabolism, with the exception of PDGF receptors (p < 0.05), when compared with the osteoblasts obtained from the normal frontal bone. These results support a hypothesis in which a complex cell signaling mechanism regulates morphogenesis of the cranial vault at the sutural sites rather than a set of biomechanical tension forces that are exerted by the underlying brain.

Interferon-responsive protein kinase (p68) and proliferating cell nuclear antigen are inversely distributed in head and neck squamous cell carcinoma.

PKR (protein kinase, interferon-responsive) is a ribosomal-associated protein kinase found in all human cells. When activated by dsRNA or polyanionic substances, PKR efficiently inhibits cellular protein synthesis. PKR expression has been correlated with cellular differentiation in a number of tumor types, including squamous cell carcinoma of the head and neck region. Although transfection of PKR into mouse fibroblasts and yeast cells inhibits proliferation, it is not known if modulation of native PKR levels occurs during cellular proliferation and differentiation in human normal and neoplastic tissues. To determine whether PKR expression was inversely related to proliferative activity in vivo, we used double-label immunohistochemistry to colocalize PKR and the proliferation marker, proliferating cell nuclear antigen (PCNA), in a series of head and neck squamous cell carcinomas. Overall, neoplasms demonstrating high levels of PKR showed low levels of PCNA immunoreactivity; carcinomas with low levels of PKR expressed high levels of PCNA. Within individual tumors, PKR and PCNA showed an inverse regional distribution: PKR was located predominantly in the center of tumor nests, while PCNA was restricted to the periphery. Patients whose tumors expressed high levels of both PKR and PCNA had the longest mean disease-free survival. These findings support the hypothesis that PKR levels are modulated in cell proliferation and differentiation in head and neck squamous cell carcinoma. Further studies are needed to clarify the mechanisms underlying the antiproliferative activity of PKR.

Expression of p68 protein kinase and its prognostic significance during IFN-alpha therapy in patients with carcinoid tumours.

The aim of this study was to evaluate the antiproliferative effects of interferon alpha (IFN-alpha) on neuroendocrine differentiated cell lines and, retrospectively, to assess the prognostic significance of p68 protein kinase (PKR) induction in neuroendocrine gut and pancreatic tumour patients. Archive specimens from 56 patients were studied, 43 before IFN-alpha and 56 during therapy. The tissues were immunostained for p68 protein kinase (PKR) using the monoclonal antibody (MAb) TJ4C4. A significant increase in immunostaining after treatment with IFN-alpha compared with before treatment (3.47 +/- 0.12 versus 2.72 +/- 0.15, P < 0.001) was noted. The p68 score was significantly increased after treatment only in patients with stable disease before = 2.71 +/- 0.19, after = 3.40 +/- 0.14 (P < 0.001) or an objective response before 3.13 +/- 0.22, after = 4.00 +/- 0.24 (P < 0.05) but not in those with progressive disease (before = 2.32 +/- 0.24, after 2.86 +/- 0.26, NS). A low p68 score (< 3.0) during treatment was a predictor of shorter duration of response and overall survival (P = 0.0062 and P < 0.0001, respectively). Furthermore, IFN-alpha showed a significant antiproliferative effect (by [3H]thymidine incorporation) on two carcinoid tumour cell lines in a dose-dependent manner which correlated with a dose-dependent induction of p68 mRNA and protein expression (by Northern and Western blot analysis). We conclude that IFN-alpha can effectively inhibit the in vitro growth of carcinoid tumor cell lines and upregulates the expression of p68 at both mRNA and protein levels in carcinoid tumours. The induction of p68 could be a prognostic indicator of response in patients with carcinoid tumours during IFN-alpha treatment.

Cytoplasmic localization of endothelial constitutive nitric oxide synthase in endometrial carcinomas.

BACKGROUND: Production of nitric oxide by nitric oxide synthase (NOS) has been implicated in numerous physiologic and pathophysiologic processes including mutagenesis. This study was designed to examine the expression of the endothelial constitutive isoform of NOS (ecNOS) in endometrial carcinomas. METHODS: Fifty endometrial carcinomas (42 endometrioid, 4 serous papillary, 2 clear cell, and 2 adenosquamous carcinomas) and 21 normal endometrial gland tissue specimens (5 cases of proliferative, 5 early secretory, 5 mid-secretory, and 5 late secretory and 1 menstrual phase endometrium), previously formalin fixed and paraffin embedded, were immunostained using a commercially available anti-ecNOS monoclonal antibody. Localization of ecNOS staining to the plasma membrane, cytoplasm and nuclei was graded with respect to overall staining intensity (0-3+ scale) and frequency (percentage of immunoreactive cells). RESULTS: Relatively little staining for ecNOS was localized to the plasma membrane in either normal or neoplastic tissues. Normal and hyperplastic endometrial glands demonstrated moderate cytoplasmic and weak nuclear staining in a small percentage of cells. While ecNOS expression was most prominent in epithelial cells, weak expression was also rarely noted in endometrial stroma, blood vessel walls, and endothelium. We found a broad range of ecNOS expression in endometrial carcinomas, predominantly localized to the cytoplasm and nuclei. No statistically significant difference in ecNOS staining frequency or intensity was found between different histologic subtypes of endometrial carcinomas. No apparent correlation was found between ecNOS expression and tumor stage, grade, extension to the lower uterine segment or cervix, nodal or distant metastases, recurrence, or final patient status among patients with endometrioid adenocarcinomas. Endometrioid tumors invading more than 1/2 of myometrial thickness (n = 18) had significantly higher cytoplasmic staining intensity than those tumors limited to the inner 1/2 of myometrium (n = 27; 2.0 vs. 1.3, p < 0.04). Furthermore, a trend toward shorter disease-free survival was noted with increased staining intensity and decreased staining frequency. CONCLUSIONS: Cytoplasmic and nuclear expression of ecNOS, which is primarily limited to the glandular elements of normal endometrium, is also found to be expressed in endometrial carcinoma. Increased ecNOS staining intensity and decreased frequency tends to correlate with decreased disease-free survival. Lastly, increased cytoplasmic ecNOS staining intensity correlates with increased myometrial invasion.

Stimulation of megakaryocytopoiesis in mice by human modified C-reactive protein (mCRP)

The prototypic human acute phase reactant, C-reactive protein (CRP), and a structurally modified form of CRP (mCRP) were studied as agents which could stimulate thrombopoiesis in both in vitro and in vivo mouse models. mCRP, but not the widely studied (native) pentameric form of CRP, demonstrated significant megakaryocyte colony-stimulating activity. This activity was measured in plasma clot cultures incubated with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). mCRP increased the number of mouse megakaryocyte colonies in a dose-dependent manner. While significantly more colonies were observed in mCRP-treated cultures compared to controls, the kinetics of megakaryocyte growth and maturation were similar to those measured in cultures stimulated with PWM-SCM lacking mCRP. A low level of megakaryocyte growth-promoting activity was noted when mCRP was added to plasma clot cultures not incubated with spleen cell conditioned medium. However, the most striking activity of mCRP was in potentiating stimulated megakaryocyte colony formation (i.e., as a Meg-POT factor). In in vivo experiments, mCRP injected subcutaneously into normal mice resulted in significant increases in blood platelet numbers compared to control mice receiving sham injections. These results suggest that a modified form of CRP has thrombopoietic activity in both in vitro and in vivo mouse models, Therefore, one important biological role for CRP during an acute-phase response might be to contribute, after a structural modification, to the hematopoietic regulation of blood platelets.

Expression of the double-stranded RNA-dependent protein kinase (p68) in human breast tissues.

P68 is a potent inhibitor of protein synthesis in virally infected cells and has been suggested to function in noninfected cells as a tumor suppressor gene. We have previously demonstrated that p68 expression correlates directly with cellular differentiation and inversely with proliferative activity in normal epithelium and in several human tumor systems. In order to determine the role of p68 in human breast cancer, we utilized immunohistochemistry and mapped the expression of p68 in tissue from 200 breast biopsy specimens. A total of 434 foci, ranging from normal breast tissue to infiltrating carcinoma were examined. We found that p68 was present at basal levels in normal lobular and luminal ductal epithelial cells, with higher levels present in myoepithelial cells. Nonproliferative fibrocystic lesions showed variable expression of p68, with high levels seen within foci of apocrine metaplasia and low levels in cystically dilated terminal duct units. Low levels of p68 were seen in typical ductal proliferations, lobular neoplasia (atypical lobular hyperplasia and lobular carcinoma in situ), and in fibroadenomas. Foci of atypical ductal hyperplasia in situ and invasive ductal carcinoma generally showed higher levels of p68 expression. Among the infiltrating carcinomas, p68 expression correlated with nuclear grade. This suggests that the ability of p68 to inhibit cellular proliferation may be impaired in breast cancer and that its expression, although modestly paralleling cellular differentiation, is not a predictive indicator of improved survival.