Circulating Cell-free miRNA Expression and its Association with Clinicopathologic Features in Inflammatory and Non- Inflammatory Breast Cancer.

Recent discovery showing the presence of microRNAs (miRNAs) in the circulation sparked interest in their use as potential biomarkers. Our previous studies showed the diagnostic potential of miR-451 as a serological marker for inflammatory breast cancer (IBC), miR-337- 5p and miR-30b for non-inflammatory breast cancer (non-IBC). The aim of this study is to investigate the prognostic values of circulating miRNAs by comparing the amounts of 12 circulating miRNAs in the serum of IBC and non-IBC from Tunisian breast cancer patients, and by determinating whether correlated pairs of miRNAs could provide useful information in the diagnosis of IBC and non-IBC patients. TaqMan qPCR was performed to detect circulating expression of miRNAs in serum of 20 IBC, 20 non-IBC and 20 healthy controls. Nonparametric rank Spearman rho correlation coefficient was used to examine the prognostic value of miRNAs and to assess the correlation profile between miRNAs expression. Further, a large number of miRNAs were highly correlated (rho>0.5) in both patients groups and controls. Also, the correlations profiles were different between IBC, non-IBC and healthy controls indicating important changes in molecular pathways in cancer cells. Our results showed that miR-335 was significantly overexpressed in premenopausal non-IBC patients; miR-24 was significantly overexpressed in non-IBC postmenopausal patients. Patients with previous parity had higher serum of miR-342-5p levels than those without. Furthermore, patients with HER2+ IBC present lower serum levels of miR-15a than patients with HER2- disease. Together, these results underline the potential of miRNAs to function as diagnostic and prognostic markers for IBC and non-IBC, with links to the menopausal state, Her2 status and parity.

Strengthening cancer biology research, prevention, and control while reducing cancer disparities: student perceptions of a collaborative master's degree program in cancer biology, preventions, and control.

This article provides the findings of a survey of previous and current students in the UDC/GU-LCCC master's degree program. This master's degree program, Cancer Biology, Prevention, and Control is administered and taught jointly by faculty of a Minority Serving Institution, the University of the District of Columbia, and the Lombardi Comprehensive Cancer Center to incorporate the strengths of a community-based school with a research intensive medical center. The program was initiated in 2008 through agreements with both University administrations and funding from the National Cancer Institute. The master's degree program is 36 credits with a focus on coursework in biostatistics, epidemiology, tumor biology, cancer prevention, medical ethics, and cancer outreach program design. For two semesters during the second year, students work full-time with a faculty person on a laboratory or outreach project that is a requirement for graduation. Students are supported and encouraged to transition to a doctoral degree after they obtain the master's and many of them are currently in doctorate programs. Since the inception of the program, 45 students have initiated the course of study, 28 have completed the program, and 13 are currently enrolled in the program. The survey was designed to track the students in their current activities, as well as determine which courses, program enhancements, and research experiences were the least and most useful, and to discern students' perceptions of knowledge acquired on various aspects of Cancer Biology Prevention, and Control Master's Program. Thirty of the 35 individuals to whom email requests were sent responded to the survey, for a response rate of 85.7%. The results of this study will inform the strengthening of the Cancer Biology program by the Education Advisory Committee. They can also be used in the development of comparable collaborative master's degree programs designed to address the significant disparities in prevalence of cancer, low screening awareness, and access to and outcomes of cancer prevention and treatment services. This, in turn, will contribute to the elimination of the dearth of underrepresented minority scientists who address these disparities. By far, the students were satisfied with the program and believe that it has had significant impact on their ability to contribute to cancer prevention and control. They provided both general and specific recommendations to strengthen the program.

Correlation of amplification and overexpression of the c-myc oncogene in high-grade breast cancer: FISH, in situ hybridisation and immunohistochemical analyses.

In this study, we analysed gene amplification, RNA expression and protein expression of the c-myc gene on archival tissue specimens of high-grade human breast cancer, using fluorescent in situ hybridisation (FISH), nonradioactive in situ hybridisation and immunohistochemistry. The specific question that we addressed was whether expression of c-Myc mRNA and protein were correlated with its gene copy amplification, as determined by FISH. Although c-Myc is one of the most commonly amplified oncogenes in human breast cancer, few studies have utilised in situ approaches to directly analyse the gene copy amplification, RNA transcription and protein expression on human breast tumour tissue sections. We now report that by using the sensitive FISH technique, a high proportion (70%) of high-grade breast carcinoma were amplified for the c-myc gene, irrespective of status of the oestrogen receptor. However, the level of amplification was low, ranging between one and four copies of gene gains, and the majority (84%) of the cases with this gene amplification gained only one to two copies. Approximately 92% of the cases were positive for c-myc RNA transcription, and essentially all demonstrated c-myc protein expression. In fact, a wide range of expression levels were detected. Statistically significant correlations were identified among the gene amplification indices, the RNA expression scores and protein expression scores. c-myc gene amplification, as detected by FISH, was significantly associated with expression of its mRNA, as measured by the intensity of in situ hybridisation in invasive cells (P=0.0067), and by the percentage of invasive cells positive for mRNA expression (P=0.0006). c-myc gene amplification was also correlated with the percentage of tumour cells which expressed high levels of its protein, as detected by immunohistochemistry in invasive cells (P=0.0016). Thus, although multiple mechanisms are known to regulate normal and aberrent expression of c-myc, in this study, where in situ methodologies were used to evaluate high-grade human breast cancers, gene amplification of c-myc appears to play a key role in regulating expression of its mRNA and protein.

Human APC2 localization and allelic imbalance.

A second adenomatous polyposis coli (APC)-like gene, APC2/APCL, was recently described and localized to chromosome 19. We have fine mapped APC2 to a small region of chromosome 19p13.3 containing markers D19S883 and WI-19632, a region commonly lost in a variety of cancers, particularly ovarian cancer. Interphase fluorescence in situ hybridization analysis revealed an APC2 allelic imbalance in 19 of 20 ovarian cancers screened and indicates that APC2 could be a potential tumor suppressor gene in ovarian cancer. When overexpressed in SKOV3 ovarian cancer cells, which express low levels of APC2, exogenous APC2 localized to the Golgi apparatus, actin-containing structures, and occasionally to microtubules. Antibodies against the NH2 terminus of human APC2 show that endogenous APC2 is diffusely distributed in the cytoplasm and colocalizes with both the Golgi apparatus and actin filaments. APC2 remained associated with actin filaments after treatment with the actin-disrupting agent, cytochalasin D. These results suggest that APC2 is involved in actin-associated events and could influence cell motility or adhesion through interaction with actin filaments, as well as functioning independently or in cooperation with APC to down-regulate beta-catenin signaling.

Chemomorphic analysis of malathion in skin layers of the rat: implications for the use of dermatopharmacokinetic tape stripping in exposure assessment to pesticides.

The dermatopharmacokinetic (DPK) method of dermal tape stripping may prove to be a valuable addition to risk assessment protocols for toxic substances as it has been for the assessment of bioequivalence and bioavailability of topical dermatologic drugs. The measurement of drug penetration into stratum corneum (SC) with respect to time is thought to be comparable with drug distribution in underlying tissues. To examine this possibility, the dermal penetration and absorption characteristics of [(14)C]malathion in the Sprague-Dawley rat was examined by three analytical techniques. [(14)C]Malathion was applied in different vehicles for 30-min and 1-h periods of exposure. Penetration into the SC was assessed by tape stripping followed by instant electronic autoradiography (IEA). Also, the (14)C activity retained in three successive 16 microm sections of the skin application site was determined by IEA and malathion was identified by Fourier transform infrared microscopy (FTIR microscopy). Absorbed [(14)C]malathion was measured in selected tissues, organs, and the residual carcass by liquid scintillation counting (LSC). Penetration into the SC followed a linear trend. The capacity of the SC reservoir for malathion amounted to approximately 1% of the dermal dose, while approximately 6% of the dose was absorbed. Results from this study support the view that LSC remains the method of choice to efficiently and reliably quantify absorption of a radiolabeled test substance. IEA offers the ability of the user to visualize the extent and profile of dermal absorption. When IEA is combined with FTIR microscopy, an effectual tool for studying the penetration of chemicals into layers of the skin emerges. The combined use of the three analytical techniques can be used to test the validity of the DPK method in hazard evaluation and exposure assessment of the organophosphorus insecticides.

Anisomastia associated with interstitial duplication of chromosome 16, mental retardation, obesity, dysmorphic facies, and digital anomalies: molecular mapping of a new syndrome by fluorescent in situ hybridization and microsatellites to 16q13 (D16S419-D16S503).

Anisomastia is a common problem among developing adolescent girls. We recently evaluated a 22-yr-old female patient who had severe anisomastia (which had been repaired by surgery), associated with moderate to severe mental retardation, a stocky body habitus with mild obesity, dysmorphic facies (prominent, upslanting palpebral fissures, beaked nose, and a prominent philtrum), webbed neck, low hairline, and severe bilateral clinodactyly of the third, fourth, and fifth fingers with acral (but not large joint) flexion contractures. A peripheral blood high resolution karyotype revealed additional chromosomal material within the long arm of chromosome 16. Densitometric analysis of amplified polymorphic sequence-tagged sites (STS) mapping to 16q suggested that the duplication is defined by the noninvolved markers D16S419 [16q12-cen, 66 centimorgan (cM) from 16p terminus] and D16S421 (16q13-q21, 84.4 cM), encompassing a maximum of 18.4 cM of genetic distance. The STS analysis showed that the duplication was on the maternally derived chromosome 16, resulting in two maternal (and one paternal) copies of that region of chromosome 16. The location was further confirmed by bacterial artificial chromosomes (BACs) that were obtained from a commercially available library, labeled, and used for fluorescence in situ hybridization. The BACs containing STSs D16S408, D16S3137, and D16S3032 (markers that correspond to 16q13) showed two regions of hybridization, indicating that these sites were duplicated, whereas a BAC containing the STS D16S512 (which corresponds to 16q21-q22) revealed one hybridization signal per 16q, indicating that the corresponding region was not involved in the duplication. The distance between the probe signals suggested a tandem duplication. We conclude that even though trisomy 16 is the most common autosomal trisomy in spontaneous abortions, few patients with unbalanced chromosome 16 abnormalities survive to adulthood; in this report we describe one such patient with an interstitial chromosome 16 duplication (at 16q13), who had a specific phenotype associated with abnormal breast size. There are clinical similarities between this patient and patients with other 16q abnormalities, although the breast findings were unique. Molecular cytogenetics, including fluorescence in situ hybridization and densitometric analysis of amplified STSs, provided useful tools for the precise mapping of the syndrome to 16q13, where the gene(s) responsible for this phenotype might be localized.

The Syk tyrosine kinase suppresses malignant growth of human breast cancer cells.

Syk is a protein tyrosine kinase that is widely expressed in haematopoietic cells. It is involved in coupling activated immunoreceptors to downstream signalling events that mediate diverse cellular responses including proliferation, differentiation and phagocytosis. Syk expression has been reported in cell lines of epithelial origin, but its function in these cells remains unknown. Here we show that Syk is commonly expressed in normal human breast tissue, benign breast lesions and low-tumorigenic breast cancer cell lines. Syk messenger RNA and protein, however, are low or undetectable in invasive breast carcinoma tissue and cell lines. Transfection of wild-type Syk into a Syk-negative breast cancer cell line markedly inhibited its tumour growth and metastasis formation in athymic mice. Conversely, overexpression of a kinase-deficient Syk in a Syk-positive breast cancer cell line significantly increased its tumour incidence and growth. Suppression of tumour growth by the reintroduction of Syk appeared to be the result of aberrant mitosis and cytokinesis. We propose that Syk is a potent modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas.

Detection of chromosomal aberrations by a whole-genome microsatellite screen.

Chromosomal aberrations are a common cause of multiple anomaly syndromes that include developmental and growth retardation. Current microscopic techniques are useful for the detection of such aberrations but have a limit of resolution that is above the threshold for phenotypic effect. We hypothesized that a genomewide microsatellite screen could detect chromosomal aberrations that were not detected by standard cytogenetic techniques in a portion of these individuals. To test this hypothesis, we performed a genomewide microsatellite screen of patients, by use of a currently available genetic-marker panel that was originally designed for meiotic mapping of Mendelian traits. We genotyped approximately 400 markers on 17 pairs of parents and their children who had normal karyotypes. By using this approach, we detected and confirmed two cases of segmental aneusomy among 11 children with multiple congenital anomalies. These data demonstrate that a genomewide microsatellite scan can be used to detect chromosomal aberrations that are not detected by microscopic techniques.

De novo mosaic add(3) characterized to be trisomy 14q31-qter using spectral karyotyping and subtelomeric probes.

We describe a 19-year-old patient with a de novo mosaic add(3) chromosome (extra material of unknown origin on the 3q). The use of spectral karyotyping and fluorescence in situ hybridization using subtelomeric probes permitted the full characterization of the cytogenetic abnormality. The additional material on 3q was found to originate from 14q31-qter. This is one of the few reported cases with trisomy 14q31-qter and is the first mosaic case.

Characterization of a supernumerary marker derived from chromosome 17 by microdissection in an adult with MR/MCA.

We describe a 38-year-old adult who has a supernumerary marker chromosome in 40% of metaphase cells which was identified by reverse in situ hybridization with a DNA probe made by microdissection to be derived from chromosome 17. The breakpoints are estimated by G-banding and fluorescence in situ hybridization (FISH) to consist of the region from 17p11.1 to proximal 17q21. The propositus displayed severe growth retardation, kyphoscoliosis, bilateral cataracts, severe calcaneovalgus deformity of the feet, dysmorphic facies, profound mental retardation, and multiple medical problems requiring ongoing medical management. These problems included a mitral valve prolapse with regurgitation, recurrent upper and lower respiratory tract infections, and severe respiratory insufficiency. The relatively long survival of this patient enabled us to describe the natural history of this rare chromosomal mutation.

Prenatal diagnosis of partial trisomy through in situ hybridization on amniocytes with whole chromosome and centromere-specific DNA probes. A case report.

BACKGROUND: DNA probes specific for whole chromosomes or portions of chromosomes can provide important information to aid the clinician in managing pregnancy and the geneticist in relaying accurate recurrence risk information to the patient. CASE: In this case, sonography was ordered because of a low fundal height in a 29-year-old primigravida at 35 weeks' gestational age; it revealed major fetal anomalies. A small supernumerary marker was seen in some cultured amniocytes. Metaphase spreads were analyzed by means of fluorescence in situ hybridization using a centromere probe specific for chromosome 22 and a whole chromosome probe for the 11 chromosome. In situ hybridization showed that the marker chromosome was a derivative of chromosome 22 with 11q material attached near the centromere. The fetal karyotype was 47,XY,+der(22) t(11;22)(q23.3;q11.2)mat. The mother was later found to be a balanced translocation carrier. CONCLUSION: It was possible to offer rapid prenatal diagnosis for this family using interphase analysis with the 22 centromere probe. The patient had chorionic villus sampling, and DNA probes were used to analyze cells directly from the biopsy. The signal representing the supernumerary marker was not observed. Karyotype analysis later showed that the fetus was normal but a translocation carrier. This report illustrates that rapid in situ hybridization can provide important information in known cases of translocation carriers.

Pharmacokinetics, chemical interactions, and toxicological risk assessment in perspective.

Chemical mixtures and multiple routes of exposure are frequently difficult problems for exposure and risk assessors. Chemicals can interact synergistically or antagonistically at a variety of physiologic and biochemical loci within target cells. Many of these interactions can be accounted for with a thorough understanding of the pharmacokinetics of the compounds in the mixture. Many pharmacokinetic processes such as metabolism and absorption can be impacted by the presence of other chemicals in the environment and diet and as a result of medication. In addition, variations between responses as a result of different exposure scenarios (route of exposure, frequency, magnitude) can sometimes result from the impacts upon the pharmacokinetics. Pharmacokinetic models, when properly formulated and tested, can be useful tools to describe and predict the magnitude of the impact of multichemical and multiroute exposures. Several examples will be used to demonstrate this potentially powerful tool and how it can impact the risk assessment process.

Gamma aminobutyric acid radioreceptor-assay a possible biomarker for human exposure to certain agrochemicals.

Cyclodiene insecticides, hexachlorocyclohexanes, pyrethroids, bicyclophosphates, the bicycloorthocarboxylate insecticides and some of their metabolites and environmental degradation products are central nervous system toxicants with high specific binding affinity to the chloride channel of the gamma-aminobutyric acid (GABA)A receptor-ionophore sites. [35S] tertiary-butylbicyclophosphorothionate (TBPS) with specific activity higher than 60 Ci/mmole has a high binding affinity to the same sites and is now commercially available and can be used to label the GABAA receptor for the development of a radioreceptor assay technique. The GABA receptor was prepared by ultra centrifugation and dialysis of brain homogenates of either cow, goat, rat or catfish. The receptor was then labeled with [35S] TBPS and the assay was conducted by measuring the displacement of radioactivity following incubation with samples containing the analytes. A radioreceptor assay protocol was developed to measure the amount of the alpha-endosulfan in blood samples. The assay was extremely sensitive, and can detect 0.2 nM of endosulfan at a level equivalent to 0.08 ppb or 8 x 10(-11) gm of endosulfan in each ml of the blood samples.

Aluminum interaction with human brain tau protein phosphorylation by various kinases.

Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl3 at 10 nM to 10 microM range activated in-vitro [gamma-32P]ATP phosphorylation of the brain (tau) tau protein in both normal human or E. coli expressed tau forms; in the presence of the kinases P34, PKP, and PKC. However, higher concentrations of ALCl3 inhibited the tau phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl3 at 100 microM to 500 microM range induced non-enzymatic phosphorylation of tau with gamma-ATP, gamma-GTP, and alpha-GTP. AlCl3 activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of tau by Al3+ was accompanied by molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the longterm neurological effect of Al3+ in human brain leading to the formation of the neurofibrillary tangles related to Alzheimer's disease.

Malathion disposition in dermally and orally treated rats and its impact on the blood serum acetylcholine esterase and protein profile.

14C-methoxy-malathion with either pure or 50% E.C. formulated malathion were applied orally or dermally at one tenth of their LD50 to two batches of male albino rats. More than 90% of 14C was released with urine after 24 hours. The rest of 14C was detected in the feces, blood, intestines, liver and kidney in a descending order. No significant 14C was detected in other organs. Comparing the oral pure and formulated malathion treatments, there was no significant variation in the rate of disposition or excretion of 14C-malathion. However, the dermal treatment revealed that the 14C-formulated malathion was released faster than the pure one in urine in the first 24 hours; while the 14C-pure malathion showed relatively higher levels in the feces and blood in the first 24 hours. In a third batch of male albino rats, the effect of the same level of dermal treatment by either pure or 50% E.C. formulated malathion on serum acetylcholine-esterase (A. Ch. E.) activity and serum protein profile was studied. The serum A. Ch. E. activity was found to be inhibited to 40% activity after 6 to 24 hours for both treatments. However, after 96 hours the serum of the pure malathion treated rats showed full recovery of A.Ch.E. activity, while the formulated malathion treated showed only 60% activity. The SDS-PAGE analysis showed a differentiation in the serum protein bands of the 48 hours exposed rats to formulated malathion which was confirmed by the scanned gel profile. The FPLC integrated chromatograms proved an initiation of a new protein band accompanied with rearrangement of the albumin and pre-albumin bands. Thus it can be concluded that, the impact on the blood serum protein profile and A. Ch. E. activity can be used as reliable criteria to detect acute toxicity of malathion and other choline-esterase inhibitors in exposed field workers. Further research is needed to elucidate the specificity and sensitivity of such criteria as biomarkers for human exposure.