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Taking into account that fructophilic lactic acid bacteria (FLAB) can play an important role in the health of honey bees and can be used as probiotics, phenotypic properties of probiotic interest of Lactobacillus kunkeei (12 strains) and Fructobacillus fructossus bacteria (2 strains), isolated from Apis mellifera gastrointestinal tract, have been studied. We have evaluated survival of tested FLAB in honey bee gut, their susceptibility to antibiotics (ampicillin, erythromycin, tylosin), cell surface hydrophobicity, auto-aggregation ability, co-aggregation with model pathogenic bacteria, biofilm formation capacity, and effect of studied FLAB, added to sucrose syrup bee diet, on longevity of honey bees. The tested FLAB exhibited good gastrointestinal tract tolerance and high antibiotic susceptibility, which are important criteria in the screening of probiotic candidates. It was also found that all FLAB studied have high cell surface hydrophobicity and fulfil next selection criterion for their use as probiotics. Symbionts of A. mellifera showed also auto- and co-aggregation capacities regarded as valuable features for biofilm formation and inhibition of pathogens adhesion to the bee gut cells. Biofilm-development ability is a desired characteristic of probiotic lactic acid bacteria. As indicated by quantitative crystal violet-stained microplate assay and confocal laser scanning microscopy imaging, all studied A. mellifera gut isolates exhibit a biofilm positive phenotype. Moreover, it was also documented, on honey bees kept in cages, that supplementation of A. mellifera sucrose diet with FLAB decreases mortality and improves significantly longevity of honey bees. Presented research showed that A. mellifera FLAB symbionts are good candidates for application as probiotics., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Arthrosis as osteoarthritis is a global problem that affects more and more people and is associated with severe chronic pain, reduced mobility and, consequently, disability. The etiology of degenerative disease is complex and depends on many factors. However, its course was not fully understood. One of the factors affecting the development of arthrosis is obesity. Obesity is a growing problem. Over the past 30 years, the number of overweight people has almost doubled. In people suffering from obesity, whose body mass index is above 30kg/m2, the risk of developing degenerative changes in articular cartilage is six times higher than the risk of developing this disease in people with normal body weight. Osteoarthritis is detected when the symptoms get worse where the changes are already at some stage. Therefore, a lot of research is currently underway to find suitable biomarkers, which would indicate the potential development of degenerative changes in the future and at the same time the possibility of inhibiting their activity. One of them may be adipokines, which are synthesized by adipose tissue and affect cartilage. In obese people, adipokines may contribute to the inflammation of the low charterer, whichaccompanies both obesity and arthrosis. These compounds can be specific biomarkers to assess the degree of progression and severity of osteoarthritis. The aim: To assess the importance of obesity and adipokines produced by adipose tissue as specific markers of arthrosis.

Shoulder arthroplasty (SA) has improved significantly over the last twenty years. It offers the effective treatment for patients with severe shoulder dysfunctions. The indicationsfor this procedure have recently expanded tremendously. However, the most common are glenohumeral osteoarthritis, inflammatory shoulder arthropathies, rotator cuff-tear arthropathy, complex fractures of the proximal humerus and osteonecrosis of a humeral head. There is range of the procedures, such as resurfacing of humeral head, anatomictotal shoulder arthroplasty, hemiarthroplasty and reverse shoulder arthroplasty. All of them could significantly improve patients quality of life. The outcomes of the shoulder arthroplasty are very satisfying in terms of pain relief and considerable improvements in shoulder function as well as in motion. However, this procedure is not so popular as knee or hip arthroplasties. The reasons for this phenomenon are not clear. The complication rate is considerably low. The most common are periprosthetic fractures, infections, implant loosening and instability. The reasonable solution is a conversion to reverse total shoulder arthroplasty. The survivorship of the prosthesis is up to 12 years, which is acceptable by patients. Long term result are still not clear. Surgeons performing SA opt for deltopectoral approach which provides good exposure of the joint also for revisions. The aim: To summarize knowledge about SA based on current literature.

Lineage plasticity has emerged as an important mechanism of treatment resistance in prostate cancer. Treatment-refractory prostate cancers are increasingly associated with loss of luminal prostate markers, and in many cases induction of developmental programs, stem cell-like phenotypes, and neuroendocrine/neuronal features. Clinically, lineage plasticity may manifest as low PSA progression, resistance to androgen receptor (AR) pathway inhibitors, and sometimes small cell/neuroendocrine pathologic features observed on metastatic biopsy. This mechanism is not restricted to prostate cancer as other malignancies also demonstrate lineage plasticity during resistance to targeted therapies. At present, there is no established therapeutic approach for patients with advanced prostate cancer developing lineage plasticity or small cell neuroendocrine prostate cancer (NEPC) due to knowledge gaps in the underlying biology. Few clinical trials address questions in this space, and the outlook for patients remains poor. To move forward, urgently needed are: (i) a fundamental understanding of how lineage plasticity occurs and how it can best be defined; (ii) the temporal contribution and cooperation of emerging drivers; (iii) preclinical models that recapitulate biology of the disease and the recognized phenotypes; (iv) identification of therapeutic targets; and (v) novel trial designs dedicated to the entity as it is defined. This Perspective represents a consensus arising from the NCI Workshop on Lineage Plasticity and Androgen Receptor-Independent Prostate Cancer. We focus on the critical questions underlying lineage plasticity and AR-independent prostate cancer, outline knowledge and resource gaps, and identify strategies to facilitate future collaborative clinical translational and basic studies in this space., (©2019 American Association for Cancer Research.)

Extracellular matrix (ECM) proteins, and most prominently, fibronectin (Fn), are routinely used in the form of adsorbed pre-coatings in an attempt to create a cell-supporting environment in both two- and three-dimensional cell culture systems. However, these protein coatings are typically deposited in a form which is structurally and functionally distinct from the ECM-constituting fibrillar protein networks naturally deposited by cells. Here, the cell-free and scalable synthesis of freely suspended and mechanically robust three-dimensional (3D) networks of fibrillar fibronectin (fFn) supported by tessellated polymer scaffolds is reported. Hydrodynamically induced Fn fibrillogenesis at the three-phase contact line between air, an Fn solution, and a tessellated scaffold microstructure yields extended protein networks. Importantly, engineered fFn networks promote cell invasion and proliferation, enable in vitro expansion of primary cancer cells, and induce an epithelial-to-mesenchymal transition in cancer cells. Engineered fFn networks support the formation of multicellular cancer structures cells from plural effusions of cancer patients. With further work, engineered fFn networks can have a transformative impact on fundamental cell studies, precision medicine, pharmaceutical testing, and pre-clinical diagnostics., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Molecular analysis of circulating tumor cells (CTCs) at single-cell resolution offers great promise for cancer diagnostics and therapeutics from simple liquid biopsy. Recent development of massively parallel single-cell RNA-sequencing (scRNA-seq) provides a powerful method to resolve the cellular heterogeneity from gene expression and pathway regulation analysis. However, the scarcity of CTCs and the massive contamination of blood cells limit the utility of currently available technologies. Here, we present Hydro-Seq, a scalable hydrodynamic scRNA-seq barcoding technique, for high-throughput CTC analysis. High cell-capture efficiency and contamination removal capability of Hydro-Seq enables successful scRNA-seq of 666 CTCs from 21 breast cancer patient samples at high throughput. We identify breast cancer drug targets for hormone and targeted therapies and tracked individual cells that express markers of cancer stem cells (CSCs) as well as of epithelial/mesenchymal cell state transitions. Transcriptome analysis of these cells provides insights into monitoring target therapeutics and processes underlying tumor metastasis.

Triple-negative breast cancer (TNBC) is the most difficult subtype of breast cancer to treat due to a paucity of effective targeted therapies. Many studies have reported that breast cancer stem cells (BCSCs) are enriched in TNBC and are responsible for chemoresistance and metastasis. In this study, we identify LRP8 as a novel positive regulator of BCSCs in TNBC. LRP8 is highly expressed in TNBC compared to other breast cancer subtypes and its genomic locus is amplified in 24% of TNBC tumors. Knockdown of LRP8 in TNBC cell lines inhibits Wnt/β-catenin signaling, decreases BCSCs, and suppresses tumorigenic potential in xenograft models. LRP8 knockdown also induces a more differentiated, luminal-epithelial phenotype and thus sensitizes the TNBC cells to chemotherapy. Together, our study highlights LRP8 as a novel therapeutic target for TNBC as inhibition of LRP8 can attenuate Wnt/β-catenin signaling to suppress BCSCs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics.

Purpose: Population-based incidence rates of breast cancers that are negative for estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2/ neu (triple-negative breast cancer [TNBC]) are higher among African American (AA) compared with white American (WA) women, and TNBC prevalence is elevated among selected populations of African patients. The extent to which TNBC risk is related to East African versus West African ancestry, and whether these associations extend to expression of other biomarkers, is uncertain., Methods: We used immunohistochemistry to evaluate estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2/ neu, androgen receptor and aldehyde dehydrogenase 1 (ALDH1) expression among WA (n = 153), AA (n = 76), Ethiopian (Eth)/East African (n = 90), and Ghanaian (Gh)/West African (n = 286) patients with breast cancer through an institutional review board-approved international research program., Results: Mean age at diagnosis was 43, 49, 60, and 57 years for the Eth, Gh, AA, and WA patients, respectively. TNBC frequency was higher for AA and Gh patients (41% and 54%, respectively) compared with WA and Eth patients (23% and 15%, respectively; P < .001) Frequency of ALDH1 positivity was higher for AA and Gh patients (32% and 36%, respectively) compared with WA and Eth patients (23% and 17%, respectively; P = .007). Significant differences were observed for distribution of androgen receptor positivity: 71%, 55%, 42%, and 50% for the WA, AA, Gh, and Eth patients, respectively ( P = .008)., Conclusion: Extent of African ancestry seems to be associated with particular breast cancer phenotypes. West African ancestry correlates with increased risk of TNBC and breast cancers that are positive for ALDH1.

Myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. Aerobic glycolysis is a hallmark of cancer. However, the link between MDSCs and glycolysis is unknown in patients with triple-negative breast cancer (TNBC). Here, we detect abundant glycolytic activities in human TNBC. In two TNBC mouse models, 4T1 and Py8119, glycolysis restriction inhibits tumor granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) expression and reduces MDSCs. These are accompanied with enhanced T cell immunity, reduced tumor growth and metastasis, and prolonged mouse survival. Mechanistically, glycolysis restriction represses the expression of a specific CCAAT/enhancer-binding protein beta (CEBPB) isoform, liver-enriched activator protein (LAP), via the AMP-activated protein kinase (AMPK)-ULK1 and autophagy pathways, whereas LAP controls G-CSF and GM-CSF expression to support MDSC development. Glycolytic signatures that include lactate dehydrogenase A correlate with high MDSCs and low T cells, and are associated with poor human TNBC outcome. Collectively, tumor glycolysis orchestrates a molecular network of the AMPK-ULK1, autophagy, and CEBPB pathways to affect MDSCs and maintain tumor immunosuppression., (Published by Elsevier Inc.)

Purpose: Chemokine receptor 1 (CXCR1) is recognized as an actionable receptor selectively expressed by breast cancer stem cells (BCSCs). Reparixin is an investigational allosteric inhibitor of chemokine receptors 1 and 2 (CXCR1/2), and demonstrates activity against BCSCs in human breast cancer xenografts. This phase Ib clinical trial examined dose, safety, and pharmacokinetics of paclitaxel plus reparixin therapy, and explored effects of reparixin on BCSCs in patients with metastatic breast cancer (MBC) (trial registration ID: NCT02001974). Experimental Design: Eligible patients had MBC and were candidates for paclitaxel therapy. Study treatment included a 3-day run-in with reparixin oral tablets three times a day, followed by paclitaxel 80 mg/m 2 /week (days 1, 8, and 15 for 28-day cycle) + reparixin tablets three times a day for 21/28 days; three dose cohorts were examined in a 3+3 dose escalation schema. Additional patients were recruited into an expansion cohort at the recommended phase II dose to further explore pharmacokinetics, safety, and biological effects of the combination therapy. Results: There were neither G4-5 adverse events nor serious adverse events related to study therapy and no interactions between reparixin and paclitaxel to influence their respective pharmacokinetic profiles. A 30% response rate was recorded, with durable responses >12 months in two patients. Exploratory biomarker analysis was inconclusive for therapy effect on BCSCs. Conclusions: Weekly paclitaxel plus reparixin in MBC appeared to be safe and tolerable, with demonstrated responses in the enrolled population. Dose level 3, 1200 mg orally three times a day, was selected for further study in a randomized phase II trial (NCT02370238). Clin Cancer Res; 23(18); 5358-65. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

Competing Interests: Disclosure of Potential Conflicts of Interest There were no conflicts of interest.

Introduction: Triple-negative breast cancer (TNBC) is more common among African American (AA) and western sub-Saharan African breast cancer (BC) patients compared with White/Caucasian Americans (WA) and Europeans. Little is known about TNBC in east Africa., Methods: Invasive BC diagnosed 1998-2014 were evaluated: WA and AA patients from the Henry Ford Health System in Detroit, Michigan; Ghanaian/west Africans from the Komfo Anokye Teaching Hospital in Kumasi, Ghana; and Ethiopian/east Africans from the St. Paul's Hospital Millennium Medical College in Addis Ababa, Ethiopia. Histopathology and immunohistochemistry for estrogen receptor (ER), progesterone receptor (PR), and HER2/neu expression was performed in Michigan on formalin-fixed, paraffin-embedded samples from all cases., Results: A total of 234 Ghanaian (mean age 49 years), 94 Ethiopian (mean age 43 years), 272 AA (mean age 60 years), and 321 WA (mean age 62 years; p = 0.001) patients were compared. ER-negative and TNBC were more common among Ghanaian and AA compared with WA and Ethiopian cases (frequency ER-negativity 71.1 and 37.1 % vs. 19.8 and 28.6 % respectively, p < 0.0001; frequency TNBC 53.2 and 29.8 % vs. 15.5 and 15.0 %, respectively, p < 0.0001). Among patients younger than 50 years, prevalence of TNBC remained highest among Ghanaians (50.8 %) and AA (34.3 %) compared with WA and Ethiopians (approximately 16 % in each; p = 0.0002)., Conclusions: This study confirms an association between TNBC and West African ancestry; TNBC frequency among AA patients is intermediate between WA and Ghanaian/West Africans consistent with genetic admixture following the west Africa-based trans-Atlantic slave trade. TNBC frequency was low among Ethiopians/East Africans; this may reflect less shared ancestry between AA and Ethiopians.

Women with African ancestry in western, sub-Saharan Africa and in the United States represent a population subset facing an increased risk of being diagnosed with biologically aggressive phenotypes of breast cancer that are negative for the estrogen receptor, the progesterone receptor, and the HER2 / neu marker. These tumors are commonly referred to as triple-negative breast cancer. Disparities in breast cancer incidence and outcome related to racial or ethnic identity motivated the establishment of the International Breast Registry, on the basis of partnerships between the Komfo Anokye Teaching Hospital in Kumasi, Ghana, the University of Michigan Comprehensive Cancer Center in Ann Arbor, Michigan, and the Henry Ford Health System in Detroit, Michigan. This research collaborative has featured educational training programs as well as scientific investigations related to the comparative biology of breast cancer in Ghanaian African, African American, and white/European American patients. Currently, the International Breast Registry has expanded to include African American patients throughout the United States by partnering with the Sisters Network (a national African American breast cancer survivors' organization) and additional sites in Ghana (representing West Africa) as well as Ethiopia (representing East Africa). Its activities are now coordinated through the Henry Ford Health System International Center for the Study of Breast Cancer Subtypes. Herein, we review the history and results of this international program at its 10-year anniversary., Competing Interests: Authors’ disclosures of potential conflicts of interest and contributions are found at the end of this article.The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc. Evelyn JiaggeNo relationship to discloseJoseph Kwaku OppongNo relationship to discloseJessica BensenhaverNo relationship to discloseFrancis AitpillahNo relationship to discloseKofi GyanNo relationship to discloseIshmael KyeiNo relationship to discloseErnest Osei-BonsuNo relationship to discloseErnest AdjeiNo relationship to discloseMichael Ohene-YeboahNo relationship to discloseKathy ToyNo relationship to discloseKaren Eubanks JacksonNo relationship to discloseMarian AkpalooNo relationship to discloseDorcas AcheampongNo relationship to discloseBeatrice AntwiNo relationship to discloseFaustina Obeng AgyemanNo relationship to discloseZainab AlassanNo relationship to discloseLinda Ahenkorah FondjoNo relationship to discloseOsei Owusu-AfriyieNo relationship to discloseRobert Newman BrewerNo relationship to discloseAmma GyamfuahNo relationship to discloseBarbara SalemNo relationship to discloseTimothy JohnsonNo relationship to discloseMax WichaConsulting or Advisory Role: AstraZeneca/MedImmune, Verastem, Paganini Biopharma, Cormorant, Roche Research Funding: Medmmune, Dompé Farmaceutici, Paganini Biopharma, VerastemSofia MerajverNo relationship to discloseCelina KleerNo relationship to discloseJudy PangNo relationship to discloseEmmanuel Amankwaa-FrempongNo relationship to discloseAzadeh StarkNo relationship to discloseFrancis AbantangaNo relationship to discloseLisa NewmanNo relationship to discloseBaffour AwuahNo relationship to disclose

Myeloid-derived suppressor cells (MDSC) contribute to immune suppression in cancer, but the mechanisms through which they drive metastatic progression are not fully understood. In this study, we show how MDSC convey stem-like qualities to breast cancer cells that coordinately help enable immune suppression and escape. We found that MDSC promoted tumor formation by enhancing breast cancer cell stem-like properties as well as by suppressing T-cell activation. Mechanistic investigations indicated that these effects relied upon cross-talk between the STAT3 and NOTCH pathways in cancer cells, with MDSC inducing IL6-dependent phosphorylation of STAT3 and activating NOTCH through nitric oxide leading to prolonged STAT3 activation. In clinical specimens of breast cancer, the presence of MDSC correlated with the presence of cancer stem-like cells (CSC) and independently predicted poor survival outcomes. Collectively, our work revealed an immune-associated mechanism that extrinsically confers cancer cell stemness properties and affects patient outcome. We suggest that targeting STAT3-NOTCH cross-talk between MDSC and CSC could offer a unique locus to improve cancer treatment, by coordinately targeting a coupled mechanism that enables cancer stemness and immune escape. Cancer Res; 76(11); 3156-65. ©2016 AACR., (©2016 American Association for Cancer Research.)

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Background: The androgen receptor (AR) is a commonly-expressed hormone receptor in breast cancer and may be a marker of response to targeted anti-androgen therapy, a particularly attractive option for triple-negative breast cancer (TNBC). Gene expression studies suggest that ARs may distinguish a luminal/AR TNBC subtype from stem cell-like subtypes. TNBC frequency is two to three times higher in African American and African breast cancers compared with White American and European breast cancers, yet little is known regarding TNBC subtypes in high-frequency African-ancestry populations. We evaluated ARs and the mammary stem cell marker aldehyde dehydrogenase 1 (ALDH1) among breast cancers from Ghana, Africa., Methods: Overall, 147 formalin-fixed, paraffin-embedded invasive breast cancers from the Komfo Anoyke Teaching Hospital in Ghana were studied at the University of Michigan, and analyzed immunohistochemically for estrogen receptor (ER), progesterone receptor (PR), HER2/neu, ALDH1, and AR expression., Results: The median age of patients was 45 years. Only 31 cases (21 %) were ER-positive, and 14 (10 %) were HER2-positive; 89 (61 %) were TNBCs. For the entire group, 44 % were AR-positive and 45 % were ALDH1-positive. ER/PR-positive tumors were more likely to be AR-positive compared with ER/PR-negative tumors (87 vs. 26 %; p < 0.0001), but there was no association between ALDH1 and AR expression. Among the TNBC cases, 45 % were ALDH1-positive and 24 % were AR-positive. ALDH1 positivity was associated with AR positivity within the subset of TNBC (36 vs. 14 %; p = 0.019)., Conclusion: We confirmed other studies showing a high frequency of TNBC in Africa. Surprisingly, ALDH1 was found to correlate with AR expression among TNBC, suggesting that novel TNBC subtypes may exist among populations with African ancestry.

Using in vitro and in vivo experimental systems and in situ analysis, we show that growth hormone (GH) is secreted locally by normal human mammary epithelial cells upon progesterone stimulation. GH increases proliferation of a subset of cells that express growth hormone receptor (GHR) and have functional properties of stem and early progenitor cells. In 72% of ductal carcinoma in situ lesions, an expansion of the cell population that expresses GHR was observed, suggesting that GH signaling may contribute to breast cancer development.

Myeloid-derived suppressor cells (MDSCs) and cancer stem cells (CSCs) are important cellular components in the cancer microenvironment and may affect cancer phenotype and patient outcome. The nature of MDSCs and their interaction with CSCs in ovarian carcinoma are unclear. We examined the interaction between MDSCs and CSCs in patients with ovarian carcinoma and showed that MDSCs inhibited T cell activation and enhanced CSC gene expression, sphere formation, and cancer metastasis. MDSCs triggered miRNA101 expression in cancer cells. miRNA101 subsequently repressesed the corepressor gene C-terminal binding protein-2 (CtBP2), and CtBP2 directly targeted stem cell core genes resulting in increased cancer cell stemness and increasing metastatic and tumorigenic potential. Increased MDSC density and tumor microRNA101 expression predict poor survival, as does decreased tumor CtBP2 expression, independent of each other. Collectively, our work identifies an immune-associated cellular, molecular, and clinical network involving MDSCs-microRNA101-CtBP2-stem cell core genes, which extrinsically controls cancer stemness and impacts patient outcome., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Breast cancers that are negative for the estrogen receptor (ER), the progesterone receptor (PR), and the HER2 (human epidermal growth factor receptor 2) marker are more prevalent among African women, and the biologically aggressive nature of these triple-negative breast cancers (TNBCs) may be attributed to their mammary stem cell features. Little is known about expression of the mammary stem cell marker aldehyde dehydrogenase 1 (ALDH1) in African women. Novel data are reported regarding ALDH1 expression in benign and cancerous breast tissue of Ghanaian women., Methods: Formalin-fixed, paraffin-embedded specimens were transported from the Komfo Anoyke Teaching Hospital in Kumasi, Ghana to the University of Michigan for centralized histopathology study. Expression of ER, PR, HER2, and ALDH1 was assessed by immunohistochemistry. ALDH1 staining was further characterized by its presence in stromal versus epithelial and/or tumor components of tissue., Results: A total of 173 women contributed to this study: 69 with benign breast conditions, mean age 24 years, and 104 with breast cancer, mean age 49 years. The proportion of benign breast conditions expressing stromal ALDH1 (n = 40, 58%) was significantly higher than those with cancer (n = 44, 42.3%) (P = .043). Among the cancers, TNBC had the highest prevalence of ALDH1 expression, either in stroma or in epithelial cells. More than 2-fold higher likelihood of ALDH1 expression was observed in TNBC cases compared with other breast cancer subtypes (odds ratio = 2.38, 95% confidence interval 1.03-5.52, P = .042)., Conclusions: ALDH1 expression was higher in stromal components of benign compared with cancerous lesions. Of the ER-, PR-, and HER2-defined subtypes of breast cancer, expression of ALDH1 was highest in TNBC., Competing Interests: Dr. Wicha has received equity in and served on the scientific advisory board for OncoMed Pharmaceuticals, and is on the scientific advisory board for Verastem. All other authors made no disclosures., (Copyright © 2012 American Cancer Society.)

Though xenografts are used extensively for drug development in breast cancer, how well xenografts reflect the breadth of primary breast tumor subtypes has not been well characterized. Moreover, few studies have compared the gene expression of xenograft tumors to the primary tumors from which they were derived. Here we investigate whether the ability of human breast tumors (n = 20) to create xenografts in immune-deficient mice is associated with breast cancer immunohistochemical (IHC) and intrinsic subtype. We also characterize how precisely the gene expression of xenografts reprises that of parent breast tumors, using hierarchical clustering and other correlation-based techniques applied to Agilent 44K gene expression data from 16 samples including four matched primary tumor-xenograft pairs. Of the breast tumors studied, 25 % (5/20) generated xenografts. Receptor and intrinsic subtype were significant predictors of xenograft success, with all (4/4) triple-negative (TN) tumors and no (0/12) HR+Her2- tumors forming xenografts (P = 0.0005). Tumor cell expression of ALDH1, a stem cell marker, trended toward successful engraftment (P = 0.14), though CDK5/6, a basal marker, did not. Though hierarchical clustering across the 500 most variable genes segregated human breast tumors from xenograft tumors, when clustering was performed over the PAM50 gene set the primary tumor-xenograft pairs clustered together, with all IHC subtypes clustered in distinct groups. Greater similarity between primary tumor-xenograft pairs relative to random pairings was confirmed by calculation of the within-pair between-pair scatter ratio (WPBPSR) distribution (P = 0.0269), though there was a shift in the xenografts toward more aggressive features including higher proliferation scores relative to the primary. Triple-negative breast tumors demonstrate superior ability to create xenografts compared to HR+ tumors, which may reflect higher proliferation or relatively stroma-independent growth of this subtype. Xenograft tumors' gene expression faithfully resembles that of their parent tumors, yet also demonstrates a shift toward more aggressive molecular features.

Background: Approximately 50% of postmenopausal women with hormone receptor-positive early stage breast cancer treated with an aromatase inhibitor (AI) develop musculoskeletal symptoms. Standard analgesics are relatively ineffective. Duloxetine is a serotonin norepinephrine reuptake inhibitor with proven efficacy for treatment of multiple chronic pain states. The authors investigated the hypothesis that duloxetine is efficacious for treatment of AI-associated musculoskeletal symptoms., Methods: The authors performed a single-arm, open-label phase 2 study of duloxetine in postmenopausal women with breast cancer who developed new or worsening pain after treatment with an AI for at least 2 weeks. Patients were treated with duloxetine for 8 weeks (30 mg for 7 days, then 60 mg daily). The primary endpoint was a 30% decrease in average pain score over 8 weeks, and secondary outcomes included change in average and worst pain, pain interference, depression, sleep quality, and hot flashes. Statistical analysis was done with t tests for paired data., Results: Twenty-one of 29 evaluable patients (72.4%) achieved at least a 30% decrease in average pain, and 18 of 23 patients (78.3%) who completed protocol-directed treatment continued duloxetine. The mean percentage reduction in average pain severity between baseline and 8 weeks was 60.9% (95% confidence interval [CI], 48.6%-73.1%), and in maximum pain severity it was 59.9% (95% CI, 47.0-72.7%). The most common adverse events were grade 1 or 2 fatigue, xerostomia, nausea, and headache., Conclusions: Duloxetine appears to be effective and well tolerated for treatment of AI-associated musculoskeletal symptoms. Future randomized, placebo-controlled studies are warranted., (Copyright © 2011 American Cancer Society.)

This perspective on Bellacosa et al. (beginning on p. 48 in this issue of the journal) discusses the important biology of microscopically normal tissues in carriers of germ-line BRCA1 or BRCA2 mutations. The work of Bellacosa et al. is an important step toward discerning which pathways may be altered when one BRCA allele is inactivated.

Smoking is the most widespread addiction and for years is a basic addiction hazard among youngsters. The beginning of nicotine addiction is occult, during time runs as habit and biological nicotine addiction. The health consequences are seen after time. Children have their first contact with cigarettes in their own families and the problem of smoking is the consequence of following adults' example. The risk of smoking addiction rises because of negative influence of background and because of the need of being approved among men of the same age. The objective of the study was the analysis of spreading of smoking among students of PMWSZ in Opole during years 2006-2009.688 students of nursery and obstetrics faculty were investigated. Among them 175 (25.4%) were smokers and 513 (74.6%) were non-smokers. More than half of smokers (61.2%) started smoking in secondary grammar school. 110 (62.9%) of them tried to overcome their addiction. These failed attempts confirm the necessity of multi-aspect anti-nicotine help given by health professionals: doctors, psychologists, addiction therapeutics and pedagogues.

Circulating tumor cells (CTC) are emerging as a powerful prognostic and predictive biomarker in several types of cancer, including breast, colon, and prostate. Studies of CTC in metastasis and further development of CTC as a biomarker in cancer have been limited by the inability to repetitively monitor CTC in mouse models of cancer. We have validated a method to enumerate CTC in blood samples obtained from living mice using a modified version of an in vitro diagnostic system for quantifying CTC in patients. Different routes of blood collection were tested to identify a method to reproducibly recover CTC from tumor-bearing mice without interference from contaminating normal murine epithelial cells. CTC are present in blood samples from mice bearing orthotopic xenografts of several different breast cancer cell lines and primary breast cancer cells from patient biopsies. We also show that this technology can be used for serial monitoring of CTC in mouse xenograft models of human breast cancer. These results establish a new method for studying CTC in mouse models of epithelial cancer, providing the foundation for studies of molecular regulation of CTC in cancer and CTC as biomarker for therapeutic efficacy.

Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within a tumor, termed cancer stem cells. Although data have been provided to support this theory in human blood, brain, and breast cancers, the identity of pancreatic cancer stem cells has not been determined. Using a xenograft model in which primary human pancreatic adenocarcinomas were grown in immunocompromised mice, we identified a highly tumorigenic subpopulation of pancreatic cancer cells expressing the cell surface markers CD44, CD24, and epithelial-specific antigen (ESA). Pancreatic cancer cells with the CD44(+)CD24(+)ESA(+) phenotype (0.2-0.8% of pancreatic cancer cells) had a 100-fold increased tumorigenic potential compared with nontumorigenic cancer cells, with 50% of animals injected with as few as 100 CD44(+)CD24(+)ESA(+) cells forming tumors that were histologically indistinguishable from the human tumors from which they originated. The enhanced ability of CD44(+)CD24(+)ESA(+) pancreatic cancer cells to form tumors was confirmed in an orthotopic pancreatic tail injection model. The CD44(+)CD24(+)ESA(+) pancreatic cancer cells showed the stem cell properties of self-renewal, the ability to produce differentiated progeny, and increased expression of the developmental signaling molecule sonic hedgehog. Identification of pancreatic cancer stem cells and further elucidation of the signaling pathways that regulate their growth and survival may provide novel therapeutic approaches to treat pancreatic cancer, which is notoriously resistant to standard chemotherapy and radiation.

Most cancers comprise a heterogenous population of cells with marked differences in their proliferative potential as well as the ability to reconstitute the tumor upon transplantation. Cancer stem cells are a minor population of tumor cells that possess the stem cell property of self-renewal. In addition, dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. This new model for cancer will have significant ramifications for the way we study and treat cancer. In addition, through targeting the cancer stem cell and its dysregulated self-renewal, our therapies for treating cancer are likely to improve.

High-dose chemotherapy (HDCT) and autologous bone marrow transplantation (BMT) is frequently used to treat patients with metastatic cancer including breast cancer and neuroblastoma. However, the bone marrow of such patients is often contaminated with tumor cells. Recently, we have found that a recombinant adenovirus vector that contains a bcl-x, minigene (a dominant negative inhibitor of the bcl-2 family), called the bcl-x(s) adenovirus, is lethal to cancer cells derived from epithelial tissues, but not to normal human hematopoietic cells. To determine the mechanism, by which this virus spares normal hematopoietic cells, we isolated normal mouse hematopoietic stem cells and infected them with an adenovirus that contains a beta-galactosidase minigene. Such cells do not express beta-galactosidase, indicating that hematopoietic stem cells do not express transgene encoded by adenovirus vectors based upon the RSV-AD5 vector system. When breast cancer cells mixed with hematopoietic cells were infected with the bcl-x(s) adenovirus, cancer cells were selectively killed by the suicide adenoviruses. Hematopoietic cells exposed to the suicide vectors were able to reconstitute the bone marrow of mice exposed to lethal doses of y-irradiation. These studies suggest that adenovirus suicide vectors may provide a simple and effective method to selectively eliminate cancer cells derived from epithelial tissue that contaminate bone marrow to be used for autologous BMT. We therefore propose to initiate a phase I clinical trial to test the safety of this virus in women with breast cancer undergoing high does chemotherapy and autologous BMT.

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER(+) human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER(-) cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1a-deleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.

Purpose: Medical care for clinical trials is often not reimbursed by insurers, primarily because of concern that medical care as part of clinical trials is expensive and not part of standard medical practice. In June 2000, President Clinton ordered Medicare to reimburse for medical care expenses incurred as part of cancer clinical trials, although many private insurers are concerned about the expense of this effort. To inform this policy debate, the costs and charges of care for patients on clinical trials are being evaluated. In this Association of American Cancer Institutes (AACI) Clinical Trials Costs and Charges pilot study, we describe the results and operational considerations of one of the first completed multisite economic analyses of clinical trials., Methods: Our pilot effort included assessment of total direct medical charges for 6 months of care for 35 case patients who received care on phase II clinical trials and for 35 matched controls (based on age, sex, disease, stage, and treatment period) at five AACI member cancer centers. Charge data were obtained for hospital and ancillary services from automated claims files at individual study institutions. The analyses were based on the perspective of a third-party payer., Results: The mean age of the phase II clinical trial patients was 58.3 years versus 57.3 years for control patients. The study population included persons with cancer of the breast (n = 24), lung (n = 18), colon (n = 16), prostate (n = 4), and lymphoma (n = 8). The ratio of male-to-female patients was 3:4, with greater than 75% of patients having stage III to IV disease. Total mean charges for treatment from the time of study enrollment through 6 months were similar: $57,542 for clinical trial patients and $63,721 for control patients (1998 US$; P =.4), Conclusion: Multisite economic analyses of oncology clinical trials are in progress. Strategies that are not likely to overburden data managers and clinicians are possible to devise. However, these studies require careful planning and coordination among cancer center directors, finance department personnel, economists, and health services researchers.

Objectives: To determine whether a Bcl-x(S) adenoviral vector has therapeutic potential in an ascites model of human breast cancer in nude mice., Methods: Advanced ascites were developed by injecting mice intraperitoneally (IP) with MDA-MB-231 human breast carcinoma cells. Mice received sequential IP injections of the Bcl-x(S) virus or a control lac-Z adenovirus. A third group of mice received no virus. Tumor burden and survival were monitored. Histopathology and necropsies were performed on mice., Results: A single injection of the Bcl-x(S) adenovirus produced no systemic or local toxicity and no abnormal histopathology in normal mice. However, abdominal organs within these mice were transduced with the Bcl-x(S) vector. Adenoviral gene transduction efficiency in MDA-MB-231 ascites was 36+/-6.40% (n = 3). Percent weight change differences revealed that ascites bearing mice injected three times with the Bcl-x(S) vector showed a statistically significant decrease in tumor burden compared with lac-Z-injected mice (n = 7; P = .012 on days 10-15 after the first injection). Mice injected with the Bcl-x(S) vector had significantly greater survival relative to lac-Z-injected mice (n = 7; P = .0004). Bcl-x(S) protein expression was detected in aspirates of mice injected with the Bcl-x(S) vector but not the lac-Z vector. Necropsies revealed that ascites bearing mice injected with Bcl-x(S) vector lacked carcinoma in the peritoneal cavity compared with control mice., Conclusion: The Bcl-x(S) adenovirus can reduce tumor burden and increase survival in an ascites model of advanced stage breast cancer.

Oncogenes which drive the cell cycle, such as c-myc, can sensitize cells to apoptosis. This suggests the possibility that the expression of genes such as bcl-2 or bcl-xL is required to inhibit apoptosis induced by oncogene expression. We hypothesized that inhibition of Bcl-2/Bcl-xL by the pro-apoptotic Bcl-xS protein, would result in selective induction of apoptosis in mammary carcinoma cells compared to their nontransformed counterparts. Therefore, we compared the effects of Bcl-xS expression delivered by a bcl-xS adenovirus (bcl-xS-Adv) vector, on viability and apoptosis of nontransformed versus transformed mammary epithelial cells. We report that c-myc-transformed murine mammary cells are extremely sensitive to apoptosis induced by the bcl-xS adenovirus (bcl-xS-Adv) vector, whereas immortalized, nontransformed murine mammary cells are relatively resistant to apoptosis induced by this vector. Likewise, human mammary epithelial cells transduced with c-erbB-2 were more sensitive to apoptosis induced by the bcl-xS vector than the nontransformed parental cells. Similar results were obtained when we tested the effects of bcl-xS adenoviral infection on primary normal human mammary epithelial cells and SUM-190 PT cells, (a c-erbB-2 over-expressing human mammary carcinoma cell line) grown on Matrigel. These data are consistent with the hypothesis that inhibition of Bcl-2/Bcl-xL can result in selective killing of cancer cells compared to their nontransformed counterparts.

We examined the effects of all-trans retinoic acid (ATRA) and lutein (a nonprovitamin A carotenoid), on apoptosis and chemosensitivity in primary normal human mammary epithelial cells, SV40 transformed mammary cells, and MCF-7 human mammary carcinoma cells. ATRA and lutein selectively induced apoptosis in transformed but not normal human mammary cells. In addition, both compounds protected normal cells, but not transformed cells, from apoptosis induced by the chemotherapy agents etoposide and cisplatin. Furthermore, lutein and ATRA selectively increased the ratio of Bcl-xL:Bax protein expression in normal cells but not transformed mammary cells, suggesting a possible mechanism for selective modulation of apoptosis. The differential effects of lutein and ATRA on apoptotic pathways in normal versus transformed mammary epithelial cells may have important implications for chemoprevention and therapy.

Preclinical and in vitro studies have determined that copper is an important cofactor for angiogenesis. Tetrathiomolybdate (TM) was developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. Given the potency and uniqueness of the anticopper action of TM and its lack of toxicity, we hypothesized that TM would be a suitable agent to achieve and maintain mild copper deficiency to impair neovascularization in metastatic solid tumors. Following preclinical work that showed efficacy for this anticopper approach in mouse tumor models, we carried out a Phase I clinical trial in 18 patients with metastatic cancer who were enrolled at three dose levels of oral TM (90, 105, and 120 mg/day) administered in six divided doses with and in-between meals. Serum ceruloplasmin (Cp) was used as a surrogate marker for total body copper. Because anemia is the first clinical sign of copper deficiency, the goal of the study was to reduce Cp to 20% of baseline value without reducing hematocrit below 80% of baseline. Cp is a reliable and sensitive measure of copper status, and TM was nontoxic when Cp was reduced to 15-20% of baseline. The level III dose of TM (120 mg/ day) was effective in reaching the target Cp without added toxicity. TM-induced mild copper deficiency achieved stable disease in five of six patients who were copper deficient at the target range for at least 90 days.

Bcl-x(L), a prosurvival member of the Bcl-2 family that is expressed in many tumors, represses apoptosis induced by chemotherapeutic drugs in vitro. However, the contribution of apoptosis and prosurvival Bcl-2-related proteins to chemotherapy resistance in vivo is unknown and has been challenged by recent results with clonogenic survival assays. To test the ability of Bcl-x(L) to provide chemotherapy resistance to tumors, we transfected the mouse bcl-x(L) gene into the tumorigenic SCK mammary cell line and assessed the response of tumor cells to chemotherapeutic drugs in clonogenic assays and in a syngeneic mouse model. Bcl-x(L) conferred protection on SCK cells against methotrexate at certain drug concentrations, but not at all against 5-fluorouracil in clonogenic survival assays in vitro. Injection of SCK cells transfected with Bcl-x(L) or control plasmid in the mammary fat pads of syngeneic recipient mice resulted in tumors of similar size. However, although the volume of control tumors regressed up to 80% after 4 to 5 days of chemotherapy, SCK tumors expressing Bcl-x(L) did not regress and continued to grow in the presence of methotrexate or 5-fluorouracil. In addition, numbers of apoptotic cells were significantly higher in control tumors as compared to Bcl-x(L)-expressing tumors in animals treated with methotrexate or 5-fluorouracil. These results provide evidence that inhibition of apoptosis through Bcl-x(L) overexpression can promote resistance to chemotherapy in tumors in vivo.

Didemnin B (DB) is one member of a class of natural cyclic depsipeptides that display potent cytotoxicity in vitro. The detailed mechanism of action of DB is unknown, although it appears to involve the inhibition of protein biosynthesis. Additional activities of DB have established DB as a rapid and potent inducer of apoptosis in HL-60 cells. Our aim was to determine if the induction of apoptosis by DB is mediated through inhibition of protein synthesis in MCF-7 human breast carcinoma cells. Apoptosis was observed only at > or = 100 nM DB, even though inhibition of protein synthesis occurred at much lower DB concentrations (IC50 = 12 nM). DB-induced apoptosis was mediated by caspase activation, since cleavage of the caspase substrate poly(ADPribose) polymerase was observed as early as 6 hr after DB exposure. Two additional protein synthesis inhibitors, cycloheximide (CHX) and emetine (ET), failed to induce apoptosis at concentrations that completely inhibited protein synthesis. Moreover, DB-induced apoptosis was enhanced only slightly by pre- and co-treatment with CHX and ET. Thus, inhibition of protein synthesis alone was not sufficient to induce apoptosis in these cells. As a measure of antiproliferative potential, DB (1-5 nM) inhibited the colony forming ability of MCF7 cells regardless of pretreatment with CHX. In conclusion, additional effects of DB, independent of protein synthesis inhibition, are proposed to account for its ability to induce apoptosis and prevent cell proliferation.

Dietary factors may influence the risk for breast cancer and also the prognosis following diagnosis and treatment. The aim of this study was to assess whether self-reported prediagnosis diet or other patient factors associated with breast cancer incidence were predictive of recurrence and survival. Patients (n = 149) diagnosed with primary breast cancer between 1989 and 1991 were followed for five or more years. Total energy (hazard ratio (HR) = 1.58, 95%, confidence interval (CI) = 1.05, 2.38) as well as total (HR = 1.46, 95% CI = 1.05, 2.01), saturated (HR = 1.79, 95% CI = 1.05, 3.04), and monounsaturated (HR = 1.65, 95% CI = 1.09, 2.49) fat intakes were associated with increased risk, and energy-adjusted bread and cereal consumption (HR = 0.55, 95% CI = 0.33, 0.93) with decreased risk of recurrence. Both total energy (HR = 1.58, 95% CI = 1.03, 2.43) and polyunsaturated fat (HR = 1.84, 95% CI = 1.09, 3.13) intakes were associated with an increased risk of death. All associations between dietary fat and recurrence and survival attenuated following energy adjustment. Oral contraceptive use (HR = 1.28, 95% CI = 1.03, 1.60), lymph node positive status (HR = 2.36, 95% CI = 1.01, 5.49), and tumor stage (HR = 2.22, 95% CI = 1.02, 4.81) were associated with increased risk of recurrence. Tumor stage (HR = 4.96, 95% CI = 1.86, 13.23), lymph node positive status (HR = 3.31, 95% CI = 1.38, 7.95), and estrogen receptor negative status (HR = 2.46, 95% CI = 1.02, 5.94) were associated with increased risk, and arm muscle circumference (HR = 0.27, 95% CI = 0.09, 0.86) and mammographic utilization (HR = 0.77, 95% CI = 0.61, 0.98) with decreased risk of death. Higher levels of energy, fat intakes, and selected patient characteristics (particularly disease stage and anthropometric indicators of adiposity) appear to increase risk of recurrence and/or shortened survival following the diagnosis of breast cancer.

Tumor metastasis is the main cause of mortality and treatment failure in cancer patients. It is a complex biological process regulated by alternations in expression of many genes. The p53 tumor suppressor gene has been shown to regulate expression of some metastasis-related genes. p53 transcriptionally activates expression of the genes encoding epidermal growth factor receptor, matrix metalloproteinase (MMP)-2, cathepsin D, and thrombospondin-1 but represses expression of the genes encoding basic fibroblast growth factor and multidrug resistance-1. Decreased expression of E-cadherin is associated with p53 alternations. Because these p53-regulatory genes either promote or inhibit tumor metastasis, the net effect of p53 expression on tumor metastasis depends upon the pattern of expression of these genes in a particular tumor. Because radiotherapy has been shown to increase tumor metastasis in both animal and human studies and because p53 is activated by radiation or DNA-damaging reagents, here we propose the working hypothesis that p53 may promote tumor metastasis upon induction by local radiotherapy or chemotherapy in some tumor types. For patients whose tumors contain wild-type p53, MMP inhibitors might be given with or before radiotherapy or chemotherapy to prevent an increase in tumor metastasis. Special caution should be taken with patients with cancers such as nasopharyngeal carcinoma in which p53 mutation is infrequent and radiotherapy is the main choice of treatment. To test our hypothesis, three studies are proposed and could serve as an initial step in understanding the complex biological process following radiation-induced p53 activation and its roles in regulation of tumor metastasis.

Replication-deficient viral vectors are currently being used in gene transfer strategies to treat cancer cells. Unfortunately, viruses are limited in their ability to diffuse through tissue. This makes it virtually impossible to infect the majority of tumor cells in vivo and results in inadequate gene transfer. This problem can be addressed by allowing limited viral replication. Limited viral replication facilitates greater penetration of virions into tissue and can improve gene transfer. We have developed a strategy of limited viral replication using AdRSVlaclys, a chemically modified E1-deleted adenovirus, to codeliver an exogenous plasmid encoding the adenovirus E1 region. This system allows one round of viral replication. We examined the effect of this limited adenovirus replication in vitro and in vivo. In culture, codelivery of virus and pE1 resulted in a large increase in infected cells when compared with control cells exposed to virus and pUC19. In experiments on nude mice bearing HeLa ascites tumors, intraperitoneal injection of AdRSVlaclys/pE1 resulted in a significantly higher percentage of infected HeLa cells as compared with the PBS controls (p < 0.05) or the AdRSVlaclys/pUC19 controls (p < 0.01). These data demonstrate that the transcomplementation of replication-deficient adenovirus with exogenous E1 DNA leads to limited replication, and this controlled replication enhances gene transfer efficiency of adenovirus in vivo.

Noninvasive axillary lymph node staging was investigated using [131I]murine monoclonal antibody B72.3 in 16 patients with breast cancer scheduled for axillary dissection. [131I]B72.3 was injected into ipsilateral finger webs or around the breast biopsy. Scintigraphy to 72 h and gamma-counting/immunohistochemistry of nodes were performed. Specific antibody uptake (%ID/g) and the ratio of specific:nonspecific antibody uptake were not significantly different in tumor-positive versus tumor-negative nodes, suggesting that [131I]B72.3 is unsuitable to discriminate axillary node tumor involvement.

Transformation is a complex cellular process that requires several genetic abnormalities. In many cases, one of these abnormalities is an inhibition of PCD, which provides a selective advantage for tumor cells. This has been recently shown in an in vivo model, where overexpression of Bcl-XL, is a crucial step in the progression from hyperplasia to neoplasia and is accompanied by a significant decrease in tumor apoptosis [56]. Frequently, overexpression of a member of the Bcl-2 family results in a block in cell death and appears to nullify many built-in cellular defense mechanisms against cancer. Such a block presents a problem because radiation and chemotherapy, standard cancer treatments, ultimately exert their effect by induction of apoptosis and would also be made less effective. Therefore, to better treat cancer it may be necessary to develop novel methods to overcome the effects of the Bcl-2 family. One way to approach this problem is to target the cause--the molecular machinery that allows a cancer cell to survive. Advances in our understanding of apoptosis has identified the Bcl-2 family as a mediator of most apoptosis pathways, including those initiated by oncogenes, tumor suppressor genes, growth factor withdrawal, and external damaging signals. Therefore, functional inhibition of Bcl-2 family members is lethal to many cancer cells. Using gene transfer technology, we can now deliver genes that accomplish this goal. Further investigation will reveal whether this translates to improved therapy in the future.

Bcl-xs is a dominant negative repressor of Bcl-2 and Bcl-xL, both of which inhibit apoptosis. We used a replication-deficient adenoviral vector to transiently overexpress Bcl-xs in MCF-7 human breast cancer cells, which overexpress Bcl-xL. Infection with this vector induced apoptosis in vitro. We then determined the effects of intratumoral injection of bcl-xs adenovirus on solid MCF-7 tumors in nude mice. Tumors injected four times with the bcl-xs adenovirus showed a 50% reduction in size. Using terminal transferase-mediated dUTP-digoxigenin nick end labeling, we observed apoptotic cells at sites of bcl-xs adenoviral injection. These experiments demonstrate the feasibility of using bcl-xs gene therapy to induce apoptosis in human breast tumors.

Many cancers overexpress a member of the bcl-2 family of inhibitors of apoptosis. To determine the role of these proteins in maintaining cancer cell viability, an adenovirus vector that expresses bcl-xs, a functional inhibitor of these proteins, was constructed. Even in the absence of an exogenous apoptotic signal such as x-irradiation, this virus specifically and efficiently kills carcinoma cells arising from multiple organs including breast, colon, stomach, and neuroblasts. In contrast, normal hematopoietic progenitor cells and primitive cells capable of repopulating severe combined immunodeficient mice were refractory to killing by the bcl-xs adenovirus. These results suggest that Bcl-2 family members are required for survival of cancer cells derived from solid tissues. The bcl-xs adenovirus vector may prove useful in killing cancer cells contaminating the bone marrow of patients undergoing autologous bone marrow transplantation.

To better understand the molecular basis of radiation-induced cell death, we studied the role of the bcl-2 oncogene and the p53 tumor suppressor gene in this process. A temperature-sensitive mutant of murine p53 (p53Val-135) and/or bcl-2 was transfected into murine erythroleukemia cells (MEL, DP16-1, which are null in p53). We demonstrate that radiation-induced cell death occurs by both p53-dependent and -independent pathways and overexpression of bcl-2 modulates both pathways. When viability was measured 24 h post-radiation, cells that had been briefly exposed to wtp53 immediately after X-ray irradiation had decreased survival as compared to unirradiated cells expressing wtp53 or X-ray irradiated DP16-1 cells. However, at later times X-ray irradiated parental DP16-1 cells also had decreased survival compared to the unirradiated control. This decrease in survival began 48 h following radiation. Bcl-2 prevented radiation-induced cell death in DP16-1 cells expressing wtp53 and delayed radiation-induced cell death in DP16-1 cells without wtp53. X-ray irradiated cells expressing wtp53 displayed microscopic and biochemical characteristics consistent with cell death due to apoptosis. DP16-1 cells which were untransfected or co-transfected with wtp53 and bcl-2 displayed characteristics of cells undergoing necrosis. These results suggest that radiation-induced cell death occurs by both p53-dependent and p53-independent pathways. The p53-dependent pathway results in cell death via apoptosis and occurs approximately 24 h following radiation. The p53-independent pathway does not appear to involve apoptosis and occurs at a later time, starting 48 h after X-ray exposure. Thus, bcl-2 protects cells from p53-dependent radiation-induced apoptotic cell death and attenuates p53-independent radiation-induced cell death.

Hemonectin (HN) is a bone marrow (BM) protein that promotes specific attachment of immature granulocytes and their precursors within the BM. We report that HN is a glycoprotein containing both mannose and galactose residues, and provide evidence that these carbohydrates mediate granulocytic cell adhesion to HN. Carbohydrate structure was determined by digoxigenin-conjugated lectin binding to HN and indicated the presence of mannose, galactose, sialic acid, and the absence of fucose-linked oligosaccharides. The role of carbohydrates in mediating cell adhesion was examined by chemical and enzymatic deglycosylation. Deglycosylation of HN with trifluoromethanesulfonic acid, which cleaves N- and O-linked oligosaccharides, inhibits 66% of cell attachment to HN, and results in an apparent decrease in molecular weight from 60 to 50 kD. Enzymatic deglycosylation with endo-B-N-acetylglucosaminidase H, which hydrolyzes specific N-linked mannose residues, inhibits 30% of cell adhesion to HN. Finally, the role of these specific sugars in hemonectin-mediated cell adhesion was confirmed with neoglycoprotein blocking. Preincubation of BM cells with mannosyl- and galactosyl-BSA probes produces a dose-dependent inhibition of cell attachment to HN, whereas fucosyl-BSA does not inhibit cell adhesion to HN. These results show that mannose and galactose partially mediate adhesion of BM granulocytes to HN.

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy. We report that Bcl-XL, which functions like Bcl-2 to inhibit apoptosis, is highly expressed in MCF-7 human breast carcinoma cells. We used Bcl-XS, a dominant negative inhibitor of Bcl-2 and Bcl-XL, to demonstrate the role of these genes in modulating chemotherapy-induced apoptosis. Bcl-XS overexpressed in MCF-7 cells by stable transfection does not affect viability by itself but induces a marked increase in chemosensitivity to VP-16 or taxol. Using an ELISA assay which quantitates DNA damage, we demonstrate that this sensitization is due to apoptosis, suggesting the therapeutic utility of targeting this pathway.