Dr. Jean J Latimer, Ph.D. - Nova Southeastern University AutoNation Cancer Institute - Director, AutoNation Institute for Breast Cancer

Dr. Jean J Latimer

Ph.D.

Nova Southeastern University AutoNation Cancer Institute

Director, AutoNation Institute for Breast Cancer

Fort Lauderdale, Florida | United States

Main Specialties: Biochemical Genetics

Additional Specialties: Breast cancer

ORCID logohttps://orcid.org/0000-0002-4882-5170

Dr. Jean J Latimer, Ph.D. - Nova Southeastern University AutoNation Cancer Institute - Director, AutoNation Institute for Breast Cancer

Dr. Jean J Latimer

Ph.D.

Introduction

Complete List of Published Work in MyBibliography:
https://www.ncbi.nlm.nih.gov/sites/myncbi/16AbQxr2orWk7/bibliography/40740488/public/?sort=date&direction=ascending

I studied transcriptional regulation in my Ph.D. at Roswell Park Cancer Institute SUNY, Buffalo. As a postdoctoral fellow I studied embryology at the Laboratory of Radiobiology UCSF with Dr. Roger Pedersen and DNA repair with Dr. James Cleaver. I have a background in performing functional assays of DNA repair. I have extended this work into the molecular analysis of individual gene expression of the Nucleotide Excision Repair pathway using the model systems established in my own laboratory at the University of Pittsburgh Medical Center. Together with Dr. Stephen Grant I have also performed functional analyses of DNA repair and somatic mutation on some rare human DNA repair disorders (shown in the list of publications below).

I believe that genomics and transcriptomics require the context of functional assays for their true interpretation.

My laboratory has developed a number of important in vitro models related to the human breast and breast cancer. My background in developmental biology and murine embryonic stem cells has allowed my laboratory to establish a tissue engineering system that involves multiple autologous cell types from the non-diseased breast. We have established 48/48 reduction mammoplasty extended explants,12 of which are from African American patients. This system culminates in an organotypic breast epithelial/myoepithelial ductal system in vitro, after one month, over a field of stromal fibroblasts. We utilize a rich serum-containing medium based upon embryonic stem cell culture called MWRI. We have published reduction mammoplasty culture work previously in Experimental Cell Research, Cell and Tissue Research, Stem Cells and PNAS.

I have served on and chaired a number of expert panels including NIH and Department of Defense (CDMRP), AIBS Rowley New York State and Komen Foundation grant review study sections. I have contributed to ground breaking reports with the Institute of Medicine and the California Breast Cancer Fund.

Primary Affiliation: Nova Southeastern University AutoNation Cancer Institute - Fort Lauderdale, Florida , United States

Specialties:

Additional Specialties:

Research Interests:

Education

Apr 1989 - Aug 1993
University of California San Francisco
Post Doctoral Fellowship
Laboratory of Radiobiology
Sep 1982 - Mar 1989
Roswell Park Cancer Institute
Ph.D.
Cellular and Molecular Biology
Sep 1978 - May 1982
Cornell University
Bachelor of Art and Sciences

Experience

Jan 1993 - Jan 2010
University of Pittsburgh Medical Center
Assistant Professor
Obstetrics and Gynecology
Jan 2016
NSU AutoNation Breast and Solid Tumor Cancer Institute
Director
Center for Collaborative Research
Jan 2011
Nova Southeastern University
Associate Professor
Pharmaceutical Sciences
Jan 2010
University of Pittsburgh Medical Center
Adjunct Faculty
Obstretrics and Gynecology

Publications

31Publications

663Reads

449Profile Views

899PubMed Central Citations

Nucleotide excision repair is a predictor of early relapse in pediatric acute lymphoblastic leukemia.

BMC Med Genomics 2018 Oct 30;11(1):95. Epub 2018 Oct 30.

Department of Pharmaceutical Sciences, College of Pharmacy, 3200 S University Drive, Fort Lauderdale, FL, 33328, USA.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12920-018-0422-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6208034PMC
October 2018
140 Reads
2.873 Impact Factor

Regulation and disregulation of mammalian nucleotide excision repair: a pathway to nongermline breast carcinogenesis.

Photochem Photobiol 2015 Mar-Apr;91(2):493-500. Epub 2014 Dec 19.

Department of Pharmaceutical Sciences, College of Pharmacy, Nova Southeastern University, Fort Lauderdale, FL.

Nucleotide excision repair (NER) is an important modulator of disease, especially in constitutive deficiencies such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation in NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals and oxidative stress. Because NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. As NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic modulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially influences response to therapy. In this study, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1111/php.12387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715878PMC
September 2015
6 Reads
4 Citations
2.266 Impact Factor

Analysis of actively transcribed DNA repair using a transfection-based system.

Authors:
Jean J Latimer

Methods Mol Biol 2014 ;1105:533-50

Department of Pharmaceutical Sciences, Nova Southeastern University, 3301 College Avenue, Fort Lauderdale-Davie, FL, 33314-7796, USA,

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can "reactivate" or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-62703-739-6_37DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301736PMC
November 2014
8 Reads
5 Citations
1.290 Impact Factor

Unscheduled DNA synthesis: the clinical and functional assay for global genomic DNA nucleotide excision repair.

Methods Mol Biol 2014 ;1105:511-32

Department of Pharmaceutical Sciences, Nova Southeastern University, 3301 College Avenue, Fort Lauderdale-Davie, FL, 33314-7796, USA,

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-62703-739-6_36DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751080PMC
November 2014
14 Reads
4 Citations
1.290 Impact Factor

WNT10B/β-catenin signalling induces HMGA2 and proliferation in metastatic triple-negative breast cancer.

EMBO Mol Med 2013 Feb 11;5(2):264-79. Epub 2013 Jan 11.

Department of Obstetrics and Gynecology, David Geffen School of Medicine at UCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA.

Wnt/?-catenin signalling has been suggested to be active in basal-like breast cancer. However, in highly aggressive metastatic triple-negative breast cancers (TNBC) the role of ?-catenin and the underlying mechanism(s) for the aggressiveness of TNBC remain unknown. We illustrate that WNT10B induces transcriptionally active ?-catenin in human TNBC and predicts survival-outcome of patients with both TNBC and basal-like tumours. We provide evidence that transgenic murine Wnt10b-driven tumours are devoid of ER?, PR and HER2 expression and can model human TNBC. Importantly, HMGA2 is specifically expressed during early stages of embryonic mammogenesis and absent when WNT10B expression is lost, suggesting a developmentally conserved mode of action. Mechanistically, ChIP analysis uncovered that WNT10B activates canonical ?-catenin signalling leading to up-regulation of HMGA2. Treatment of mouse and human triple-negative tumour cells with two Wnt/?-catenin pathway modulators or siRNA to HMGA2 decreases HMGA2 levels and proliferation. We demonstrate that WNT10B has epistatic activity on HMGA2, which is necessary and sufficient for proliferation of TNBC cells. Furthermore, HMGA2 expression predicts relapse-free-survival and metastasis in TNBC patients.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1002/emmm.201201320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569642PMC
February 2013
19 Reads
105 Citations
8.665 Impact Factor

Identification of Hydroxysteroid (17β) dehydrogenase type 12 (HSD17B12) as a CD8+ T-cell-defined human tumor antigen of human carcinomas.

Cancer Immunol Immunother 2011 Jul 16;60(7):919-29. Epub 2011 Mar 16.

Division of Basic Research, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.

Hydroxysteroid (17?) dehydrogenase type 12 (HSD17B12) is a multifunctional isoenzyme functional in the conversion of estrone to estradiol (E2), and elongation of long-chain fatty acids, in particular the conversion of palmitic to archadonic (AA) acid, the precursor of sterols and the inflammatory mediator, prostaglandin E(2). Its overexpression together with that of COX-2 in breast carcinoma is associated with a poor prognosis. We have identified the HSD17B12(114-122) peptide (IYDKIKTGL) as a naturally presented HLA-A*0201 (HLA-A2)-restricted CD8(+) T-cell-defined epitope. The HSD17B12(114-122) peptide, however, is poorly immunogenic in its in vitro ability to induce peptide-specific CD8(+) T cells. Acting as an "optimized peptide", a peptide (TYDKIKTGL), which is identical to the HSD17B12(114-122) peptide except for threonine at residue 1, was required for inducing in vitro the expansion of CD8(+) T-cell effectors cross-reactive against the HSD17B12(114-122) peptide. In IFN-? ELISPOT assays, these effector cells recognize HSD17B12(114-122) peptide-pulsed target cells, as well as HLA-A2(+) squamous cell carcinoma of the head and neck (SCCHN) and breast carcinoma cell lines overexpressing HSD17B12 and naturally presenting the epitope. Whereas growth inhibition of a breast carcinoma cell line induced by HSD17B12 knockdown was only reversed by AA, in a similar manner, the growth inhibition of the SCCHN PCI-13 cell line by HSD17B12 knockdown was reversed by E2 and AA. Our findings provide the basis for future studies aimed at developing cancer vaccines for targeting HSD17B12, which apparently can be functional in critical metabolic pathways involved in inflammation and cancer.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00262-011-1001-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408891PMC
July 2011
8 Reads
10 Citations
3.941 Impact Factor

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer.

Proc Natl Acad Sci U S A 2010 Dec 30;107(50):21725-30. Epub 2010 Nov 30.

Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-3180, USA.

The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0914772107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003008PMC
December 2010
235 Reads
39 Citations
9.809 Impact Factor

Permanently blocked stem cells derived from breast cancer cell lines.

Stem Cells 2010 Jun;28(6):1008-18

Section of Hematology/Oncology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. However, an unanticipated result was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immunocompromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4744791PMC
June 2010
17 Reads
44 Citations
6.523 Impact Factor

Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives.

Expert Rev Mol Med 2009 Feb 5;11:e5. Epub 2009 Feb 5.

University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15232, USA.

Recent studies have suggested that the pineal hormone melatonin may protect against breast cancer, and the mechanisms underlying its actions are becoming clearer. Melatonin works through receptors and distinct second messenger pathways to reduce cellular proliferation and to induce cellular differentiation. In addition, independently of receptors melatonin can modulate oestrogen-dependent pathways and reduce free-radical formation, thus preventing mutation and cellular toxicity. The fact that melatonin works through a myriad of signalling cascades that are protective to cells makes this hormone a good candidate for use in the clinic for the prevention and/or treatment of cancer. This review summarises cellular mechanisms governing the action of melatonin and then considers the potential use of melatonin in breast cancer prevention and treatment, with an emphasis on improving clinical outcomes.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1017/S1462399409000982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301735PMC
February 2009
14 Reads
88 Citations
5.152 Impact Factor

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures.

Cell Tissue Res 2008 Sep 25;333(3):461-7. Epub 2008 Jun 25.

Center for Environmental Oncology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA.

DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00441-008-0645-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301738PMC
September 2008
19 Reads
4 Citations
3.565 Impact Factor

Elevated levels of somatic mutation in a manifesting BRCA1 mutation carrier.

Pathol Oncol Res 2007 25;13(4):276-83. Epub 2007 Dec 25.

Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15232, USA.

Homozygous loss of activity at the breast cancerpredisposing genes BRCA1 and BRCA2 (FANCD1) confers increased susceptibility to DNA double strand breaks, but this genotype occurs only in the tumor itself, following loss of heterozygosity at one of these loci. Thus, if these genes play a role in tumor etiology as opposed to tumor progression, they must manifest a heterozygous phenotype at the cellular level. To investigate the potential consequences of somatic heterozygosity for a BRCA1 mutation demonstrably associated with breast carcinogenesis on background somatic mutational burden, we applied the two standard assays of in vivo human somatic mutation to blood samples from a manifesting carrier of the Q1200X mutation in BRCA1 whose tumor was uniquely ascertained through an MRI screening study. The patient had an allele-loss mutation frequency of 19.4 x 10(-6) at the autosomal GPA locus in erythrocytes and 17.1 x 10(-6) at the X-linked HPRT locus in lymphocytes. Both of these mutation frequencies are significantly higher than expected from age-matched disease-free controls (P < 0.05). Mutation at the HPRT locus was similarly elevated in lymphoblastoid cell lines established from three other BRCA1 mutation carriers with breast cancer. Our patient's GPA mutation frequency is below the level established for diagnosis of homozygous Fanconi anemia patients, but consistent with data from obligate heterozygotes. The increased HPRT mutation frequency is more reminiscent of data from patients with xeroderma pigmentosum, a disease characterized by UV sensitivity and deficiency in the nucleotide excision pathway of DNA repair. Therefore, this BRCA1-associated breast cancer patient manifests a unique phenotype of increased background mutagenesis that likely contributed to the development of her disease independent of loss of heterozygosity at the susceptibility locus.

View Article

Download full-text PDF

Source
http://dx.doi.org/PAOR.2007.13.4.0276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301730PMC
May 2008
18 Reads
10 Citations
1.806 Impact Factor

THE EFFECTS OF STRESS ON DNA REPAIR CAPACITY.

Psychol Health 2000;15(6):881-891. Epub 2007 Dec 19.

Division of Behavioral Medicine and Oncology, University of Pittsburgh Cancer Institute, 3600 Forbes Ave, Suite 405, Pittsburgh, PA 15213.

Research has shown that lymphocytes of high-distress patients have reduced DNA repair relative to that of low-distress patients and healthy controls. Furthermore, deficits in repair are associated with an increased risk of cancer. Using and academic stress model, we hypothesized that students would exhibit lower levels of Nucleotide Excision Repair (NER) during a stressful exam period when compared to a lower stress period. Participants were 19 healthy graduate level students. NER was measured in lymphocytes using the unscheduled DNA synthesis (UDS) assay with slide autoradiography. Contrary to prediction, mean values for NER significantly during the higher stress period relative to the lower stress period controlling for background differences in repair. Furthermore, lymphocytes had significantly increased repair of endogenous damage during the higher stress period. Stress appears to directly increase DNA repair. Additionally, stress may increase DNA repair indirectly by increasing damage to DNA.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1080/08870440008405589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764099PMC
December 2007
14 Reads
33 Citations
2.182 Impact Factor

Association between biomarkers of environmental exposure and increased risk of breast cancer.

Nat Rev Cancer 2006 Aug;6(8)

University of Pittsburgh Cancer Institute, Center for Environmental Oncology, 5150 Centre Avenue, Suite 435, Pittsburgh, Pennsylvania, 15232, USA.

In December 2005, Deborah Winn’s Science and Society article reviewed research findings from the Long

Island Breast Cancer Study Project (LIBCSP)1. Her conclusion that the LIBCSP provided “no evidence that

environmental exposures were responsible” for breast cancer patterns on Long Island is misleading and

understates the complexity of the data. In fact, Winn reviews three reports that show a clear association

between biomarkers of environmental exposure and increased risk of breast cancer on Long Island,

including: 1) women with the highest exposure to polycyclic aromatic hydrocarbons had a 50% increase in

breast cancer risk2; 2) those with elevated levels of a specific polychlorinated biphenyl (PCB) variant in breast

fat had a fourfold increased risk of recurrence of the disease3; 3) women who lived within 1 mile of hazardous

waste sites that contained organochlorines in Nassau and Suffolk counties had a threefold increased risk of

breast cancer4. In addition, her review also notes that women living on Cape Cod for 5 or more years had a

20% increased risk of breast cancer compared with those in the rest of the state. Interestingly, for the Cape Cod

cohort, women who had been living there for 25–30 years — since 1948, the year that the use of DDT began in

that area — had the greatest odds ratio for developing cancer5.’

Experimental research has clearly shown that many environmental exposures that cannot easily be studied

in humans can damage cells and cause disease, including cancer. It is very difficult to reconstruct real-life

exposures for a multifactoral disease like breast cancer, where the timing of exposure is uncertain and the

ability to reconstruct a lifetime exposure assessment using biomarker analysis or Geographical Information

System (GIS) mapping techniques are limited. Despite all of the epidemiological research that has been

performed, the causes of regional ‘hot spots’ for breast cancer in the United States, are not well understood at

this time.

The use of case–control studies to elucidate geographic risks of breast cancer, as was done in the LIBCSP

studies, raises the question of what are the appropriate controls. In an area of high risk, it is possible that cases

might be the most sensitive or hypersusceptible individuals, whereas local, regional controls are those who

are delayed in developing breast cancer. This idea is supported by a recent study of sisters that were

discordant for breast cancer, which found that DNA repair capacity was lower in patients with breast cancer

compared with their disease-free sisters. Women in the lowest quartile for DNA repair had nearly 3 times the

risk of breast cancer compared with their individual sisters (2.99 CI = 1.45–6.17, P = .02) (6).

In truth, a number of studies conducted as part of the LIBCSP did not find associations between the

environment and breast cancer. The absence of evidence connecting specific environmental factors and

patterns of breast cancer in some studies should not be confused with proof that such a connection does not

exist. Rather, it could reflect the methodological complexities of epidemiological studies, the difficulties of

obtaining appropriate surrogates or direct measures of relevant exposures that can take place over a lifetime,

and the dearth of reliable biomarkers for accurate correlative historical studies.

1. Winn, D. M. The Long Island Breast Cancer Study Project. Nature Rev. Cancer 5, 986–

994 (2005).

© 2006

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4764098PMC
http://dx.doi.org/10.1038/nrc1755-c1DOI Listing
August 2006
21 Reads
42.784 Impact Factor

Prospective screening study of 0.5 Tesla dedicated magnetic resonance imaging for the detection of breast cancer in young, high-risk women.

BMC Womens Health 2006 Jun 26;6:10. Epub 2006 Jun 26.

Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Background: Evidence-based screening guidelines are needed for women under 40 with a family history of breast cancer, a BRCA1 or BRCA2 mutation, or other risk factors. An accurate assessment of breast cancer risk is required to balance the benefits and risks of surveillance, yet published studies have used narrow risk assessment schemata for enrollment. Breast density limits the sensitivity of film-screen mammography but is not thought to pose a limitation to MRI, however the utility of MRI surveillance has not been specifically examined before in women with dense breasts. Also, all MRI surveillance studies yet reported have used high strength magnets that may not be practical for dedicated imaging in many breast centers. Medium strength 0.5 Tesla MRI may provide an alternative economic option for surveillance.

Methods: We conducted a prospective, nonrandomized pilot study of 30 women age 25-49 years with dense breasts evaluating the addition of 0.5 Tesla MRI to conventional screening. All participants had a high quantitative breast cancer risk, defined as > or = 3.5% over the next 5 years per the Gail or BRCAPRO models, and/or a known BRCA1 or BRCA2 germline mutation.

Results: The average age at enrollment was 41.4 years and the average 5-year risk was 4.8%. Twenty-two subjects had BIRADS category 1 or 2 breast MRIs (negative or probably benign), whereas no category 4 or 5 MRIs (possibly or probably malignant) were observed. Eight subjects had BIRADS 3 results, identifying lesions that were "probably benign", yet prompting further evaluation. One of these subjects was diagnosed with a stage T1aN0M0 invasive ductal carcinoma, and later determined to be a BRCA1 mutation carrier.

Conclusion: Using medium-strength MRI we were able to detect 1 early breast tumor that was mammographically undetectable among 30 young high-risk women with dense breasts. These results support the concept that breast MRI can enhance surveillance for young high-risk women with dense breasts, and further suggest that a medium-strength instrument is sufficient for this application. For the first time, we demonstrate the use of quantitative breast cancer risk assessment via a combination of the Gail and BRCAPRO models for enrollment in a screening trial.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1186/1472-6874-6-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1553433PMC
June 2006
13 Reads
13 Citations
1.806 Impact Factor

Haploinsufficiency for BRCA1 is associated with normal levels of DNA nucleotide excision repair in breast tissue and blood lymphocytes.

BMC Med Genet 2005 Jun 14;6:26. Epub 2005 Jun 14.

Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

Background: Screening mammography has had a positive impact on breast cancer mortality but cannot detect all breast tumors. In a small study, we confirmed that low power magnetic resonance imaging (MRI) could identify mammographically undetectable tumors by applying it to a high risk population. Tumors detected by this new technology could have unique etiologies and/or presentations, and may represent an increasing proportion of clinical practice as new screening methods are validated and applied A very important aspect of this etiology is genomic instability, which is associated with the loss of activity of the breast cancer-predisposing genes BRCA1 and BRCA2. In sporadic breast cancer, however, there is evidence for the involvement of a different pathway of DNA repair, nucleotide excision repair (NER), which remediates lesions that cause a distortion of the DNA helix, including DNA cross-links.

Case Presentation: We describe a breast cancer patient with a mammographically undetectable stage I tumor identified in our MRI screening study. She was originally considered to be at high risk due to the familial occurrence of breast and other types of cancer, and after diagnosis was confirmed as a carrier of a Q1200X mutation in the BRCA1 gene. In vitro analysis of her normal breast tissue showed no differences in growth rate or differentiation potential from disease-free controls. Analysis of cultured blood lymphocyte and breast epithelial cell samples with the unscheduled DNA synthesis assay (UDS) revealed no deficiency in nucleotide excision repair (NER).

Conclusion: As new breast cancer screening methods become available and cost effective, patients such as this one will constitute an increasing proportion of the incident population, so it is important to determine whether they differ from current patients in any clinically important ways. Despite her status as a BRCA1 mutation carrier, and her mammographically dense breast tissue, we did not find increased cell proliferation or deficient differentiation potential in her breast epithelial cells, which might have contributed to her cancer susceptibility. Although NER deficiency has been demonstrated repeatedly in blood samples from sporadic breast cancer patients, analysis of blood cultured lymphocytes and breast epithelial cells for this patient proves definitively that heterozygosity for inactivation of BRCA1 does not intrinsically confer this type of genetic instability. These data suggest that the mechanism of genomic instability driving the carcinogenic process may be fundamentally different in hereditary and sporadic breast cancer, resulting in different genotoxic susceptibilities, oncogene mutations, and a different molecular pathogenesis.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2350-6-26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1215484PMC
June 2005
17 Reads
14 Citations
2.083 Impact Factor

Analysis of DNA repair using transfection-based host cell reactivation.

Methods Mol Biol 2005 ;291:321-35

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA, USA.

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique using UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a reporter enzyme, the amount of damaged plasmid that a cell can "reactivate" or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Because it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair (TCR), a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860737PMC
February 2005
16 Reads
23 Citations
1.290 Impact Factor

Unscheduled DNA synthesis: a functional assay for global genomic nucleotide excision repair.

Methods Mol Biol 2005 ;291:303-20

Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

The unscheduled DNA synthesis (UDS) assay measures a cell's ability to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique in living cells by creating 6-4 photoproducts and pyrimidine dimers using UVC irradiation, then allowing for their repair. Repair is quantified by the amount of radioactive thymidine incorporated after this insult, and the length of time allowed for this incorporation is specific for repair of particular lesions. Radioactivity is evaluated by grain counting after autoradiography. The results are used to diagnosis repair-deficient disorders clinically and provide a basis for investigation of repair deficiency in human tissues or tumors. At the present time, no other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation is not an equivalent technique, as it specifically measures transcription-coupled repair at active genes, a subset of total NER.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4751077PMC
February 2005
17 Reads
20 Citations
1.290 Impact Factor

Unique tissue-specific level of DNA nucleotide excision repair in primary human mammary epithelial cultures.

Exp Cell Res 2003 Nov;291(1):111-21

Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA 15213, USA.

DNA repair is essential for the maintenance of genomic integrity and stability. Nucleotide excision repair (NER) is a major pathway responsible for remediation of damage caused by UV light, bulky adducts, and cross-linking agents. We now show that NER capacity is differentially expressed in human tissues. We established primary cultures of peripheral blood lymphocytes (PBLs: N = 33) and foreskin fibroblasts (FF: N = 6), as well as adult breast tissue (N = 22) using a unique culture system, and measured their NER capacity using the unscheduled DNA synthesis (UDS) functional assay. Relative to FF, primary cultures of breast cells exhibited only 24.6 +/- 2.1% of NER capacity and PBLs only 8.9 +/- 1.2%. Cells from the breast therefore have a unique and distinctive DNA repair capacity. The NER capacities of all three cell types had similar coefficients of variation in the range of 10%-15%, which should be taken into account when running controls for this contextual assay. Unlike previous studies and speculation in the field, we found that NER was not affected by cell morphology, donor age, or proliferation as measured by the S phase index. While the NER capacity of the transformed lymphoblastoid cell line TK6 was within the range of our PBL samples, the breast tumor-derived MDA MB-231 cell line was four-fold higher than normal breast tissue. These studies show that analysis of baseline DNA repair in normal human cell types is critical as a basis for evaluation of the effects of "mutator" genes as etiological factors in the development of cancer.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729389PMC
November 2003
14 Reads
23 Citations
3.246 Impact Factor

Mammography and Beyond: Developing Technologies for the Early Detection of Breast Cancer: A Non-Technical Summary

National Academies Press

 X-ray mammography screening is the current mainstay for early breast

cancer detection. It has been proven to detect breast cancer at an earlier stage and to reduce the number of women dying from the disease. However, it has a number of limitations. These current limitations in early breast cancer detection technology are driving a surge of new technological developments, from modifications of x-ray mammography such as computer programs that can indicate suspicious areas, to

newer methods of detection such as magnetic resonance imaging (MRI) or

biochemical tests on breast fluids. To explore the merits and drawbacks of these new breast cancer detection techniques, the Institute of Medicine of the National Academy of Sciences convened a committee of experts. During its year of operation, the committee examined the peer-reviewed literature, consulted with other experts in the field, and held two public workshops. In addition to identifying promising new technologies for early detection, the committee explored potential barriers that might prevent the development of newdetection methods and their common usage. Such barriers could include lack of funding from agencies that support research and lack of investment in the commercial sector; complicated, inconsistent, or unpredictable federal regulations; inadequate insurance reimbursement; and limited access to or unacceptability of breast cancer detection technology for women and their doctors. Based on the findings of their study, the committee prepared a report entitled Mammography and Beyond: Developing Technology for Early Detection of Breast Cancer,  which was published in the spring of 2001. This is a nontechnical summary of that report.


The committee concluded that a great deal of work remains to be done,

particularly in the field of cancer markers (the study of biological characteristics, or markers, associated with cancer). This area holds promise, however, for improving the accuracy of breast cancer screening, diagnosis, and prognosis. The committee noted that improved imaging technologies that allow doctors to detect  breast abnormalities at an earlier, pre-invasive stage may lead to more over-treatment of women unless the imaging procedures are coupled with molecular marker technology that can determine which abnormalities are likely to spread aggressively and become life-threatening.

The committee provided several suggestions for improving the process of

developing new technologies, including government support for the discovery and development of markers associated with breast cancer or breast cancer precursors, more consistent FDA regulations regarding approval of cancer thx detection devices, and a coordinated approval and coverage assessment scheme for screening tests. The committee also made several recommendations intended to optimize the use and benefit of the proven technologies that are currently available. Those recommendations include expanding the breast cancer screening program of the Centers for Disease Control and Prevention (CDC) for women without health insurance, studying reimbursement rates for x-ray mammography to determine whether they adequately cover the total costs of providing the procedure, and determining whether there is a current or impending shortage of radiologists trained in breast imaging.

The rationale for these recommendations is summarized in this report.


View Article
December 2001

24 Citations

Mammography and Beyond: : Developing Technologies for the Early Detection of Breast Cancer

Institute of Medicine Book

Description

Each year more than 180,000 new cases of breast cancer are diagnosed in women in the U.S. If cancer is detected when small and local, treatment options are less dangerous, intrusive, and costly-and more likely to lead to a cure.

Yet those simple facts belie the complexity of developing and disseminating acceptable techniques for breast cancer diagnosis. Even the most exciting new technologies remain clouded with uncertainty. Mammography and Beyond provides a comprehensive and up-to-date perspective on the state of breast cancer screening and diagnosis and recommends steps for developing the most reliable breast cancer detection methods possible.

This book reviews the dramatic expansion of breast cancer awareness and screening, examining the capabilities and limitations of current and emerging technologies for breast cancer detection and their effectiveness at actually reducing deaths. The committee discusses issues including national policy toward breast cancer detection, roles of public and private agencies, problems in determining the success of a technique, availability of detection methods to specific populations of women, women's experience during the detection process, cost-benefit analyses, and more.

Examining current practices and specifying research and other needs, Mammography and Beyond will be an indispensable resource to policy makers, public health officials, medical practitioners, researchers, women's health advocates, and concerned women and their families.

Preface

Breast cancer remains a leading cause of cancer death among women

in the United States. More than 180,000 new cases of invasive breast

cancer are diagnosed each year, and more than 40,000 women die of the

disease. Recent years, however, have seen improvements in survival

attributed to better treatment and earlier diagnosis. Research efforts have

been directed toward better treatment, preventive strategies, and early

detection. Although mammography has been the mainstay of early detection, its limitations are well recognized and the search for more effective technologies for early detection has been receiving increased attention. As part of this increased attention, the Institute of Medicine (IOM) convened a committee to examine the current state of the art in early breast cancer detection, to identify promising new technologies, and to examine the many steps in medical technology development and the policies that influence their adoption and use. The IOM committee consisted of a 16-member interdisciplinary group with a wide range of views and expertise in breast cancer, medical imaging, cancer biology, epidemiology, economics, and technology assessment. The committee examined the peer-reviewed literature, met four times, held two workshops that dealt with new technologies as well as policies related to their adoption and dissemination, and consulted with experts in the field.


Early detection is widely believed to save lives by facilitating intervention

early in the course of the disease, at a stage when cancer treatment

is most likely to be effective. This concept, however, belies a number

of complexities, not the least of which is the need to understand the

basic biology of breast cancer. The committee recognized the need for

research on the natural history of breast cancer to more clearly define the

significance of early lesions, the need for the development of biomarkers,

and the importance of assessing the effectiveness of new technologies in

decreasing morbidity and mortality. This report describes many novel

technologies that are being developed for the purpose of early breast

cancer detection, as well as recent technological advances in detection

modalities already in use. Because the many technologies that the committee examined were at different stages of development and thus the

evidence of their accuracy and effectiveness varied, the committee found

it difficult to predict which of the many new technologies were likely to

play a role in the future of early breast cancer detection.


The committee also identified a number of barriers to both the development and the dissemination of new technologies and made recommendations for actions that can be taken to overcome them. Many new technologiesare on the horizon and intriguing research in basic biology is under way, but much remains to be done. We are hopeful that this report will contribute in some small way to the efforts to improve our ability to detect breast cancer at an early stage. The committee was impressed with the dedication and commitment of the researchers in both the public and the private sectors and with the governmental personnel working tosave the lives of women, and we are hopeful that their efforts will prove fruitful.

View Article
December 2001

217 Citations

Preterm premature rupture of membranes without labor is not associated with increased levels of matrix metalloproteinase-9 protein.

Prenat Neonatal Med 2001 Aug;6(4):219-226

Magee-Women's Research Institute, Department of Obstetrics and Gynecology and Reproductive Sciences, School of Medicine, University of Pittsburgh, Pennsylvania, USA.

Objectives: Preterm premature rupture of membranes (PROM) accounts for 30-40% of all preterm births. The objectives of this study were to determine whether matrix metalloproteinase-9 (MMP-9) is increased in preterm PROM fetal membranes, whether labor or gestational age affects expression, and whether the increase is localized to the rupture site or is membrane-wide.

Methods: Fetal membranes were collected from 15 pregnancies complicated by preterm PROM and 26 control cases, which delivered at term or preterm without PROM. The preterm PROM cases represented both patients who labored and those who did not. Membrane samples at the rupture site and a remote site (approximately > 5 cm) were analyzed for MMP-9 protein and enzymatic activity by Western blot and gelatin zymography, respectively.

Results: MMP-9 levels in fetal membranes were similar at both the rupture and the remote sites. The highest levels of total MMP-9 protein were found in preterm PROM patients with labor ( < 0.05) and were increased four-fold over protein levels in non-laboring preterm PROM patients delivered by Cesarean section ( < 0.001). In preterm PROM patients without labor, levels of MMP-9 protein were similar to those of non-laboring patients at term and preterm. Zymography correlated with protein results in all membranes.

Conclusions: Preterm PROM without labor is not associated with increased membrane levels of MMP-9 protein, suggesting that its local elevation does not play a role in early membrane rupture.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4769030PMC
August 2001
3 Citations

Analysis of genomic instability using multiple assays in a patient with Rothmund-Thomson syndrome.

Clin Genet 2000 Sep;58(3):209-15

Department of Environmental and Occupational Health, University of Pittsburgh, PA, USA.

We report on a patient with Rothmund-Thomson syndrome (RTS) whose cytogenetic evaluation showed a normal karyotype with no evidence of trisomy mosaicism or chromosomal rearrangements. Cultured lymphocytes from the patient, her mother, and a control exposed to mitomycin C and diepoxybutane did not show increased sensitivity to the dialkylating agents. Unlike some previous reports, we found no evidence of a deficiency in nucleotide excision repair, as measured with the functional unscheduled DNA synthesis assay. Glycophorin A analysis of red blood cells for somatic mutation revealed suspiciously high frequencies of both allele loss and loss-and-duplication variants in the blood of the patient, a pattern consistent with observations in other RecQ-related human diseases, and evidence for clonal expansion of a mutant clone in the mother. Discrepant results in the literature may reflect true heterogeneity in the disease or the fact that a consistent set of tests has not been applied to RTS patients.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712958PMC
September 2000
3 Reads
25 Citations
3.931 Impact Factor

Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo.

Exp Cell Res 1996 Oct;228(1):19-28

Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143, USA.

In order to determine whether there is differential cell-type-specific DNA repair we measured the nucleotide excision repair capacity of the four distinct cell lineages that comprise the extraembryonic yolk sac using the unscheduled DNA synthesis assay. Yolk sacs from mouse embryos at 11.5-12.5 days gestation were microdissected to yield purified trophoblast, parietal endoderm, mesoderm, and visceral endoderm, as well as fetal skin fibroblasts which were then grown as primary explants. At this midgestational stage of development, the yolk sac provides essential functions for the sustenance of the embryo while the complex process of organogenesis is proceeding in the liver, kidney, and gut. Trophoblast giant cells, parietal endoderm, and visceral endoderm all demonstrated low levels of unscheduled DNA synthesis consistent with levels measured in adult mouse skin fibroblasts. As has previously been documented, embryonic mouse skin fibroblasts were reproducibly 2- to 3-fold higher than adult mouse skin fibroblasts in levels of DNA excision repair. The extraembryonic mesoderm, however, displayed a statistically significant level of unscheduled DNA synthesis 10-fold higher than adult mouse skin fibroblasts or the other lineages of the midgestation yolk sac. Further, the S-indexes of these lineages were also determined to assess the possible relevance of differential repair to the proliferative status of the cells. These data demonstrate that DNA excision repair capacity is lineage-specific during embryogenesis in the mouse. These studies may begin to provide a context for understanding the perplexing developmental aspects such as the characteristic congenital abnormalities associated with the human heritable DNA repair deficiency diseases.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1006/excr.1996.0294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729398PMC
October 1996
38 Reads
14 Citations
3.246 Impact Factor

Novel flow-cytometric method for separating cell types in differentiated F9 embryoid bodies.

Cytometry 1995 Oct;21(2):145-52

Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, USA.

The differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of alpha-fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence-activated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid-induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of alpha-fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies.

View Article

Download full-text PDF

Source
http://dx.doi.org/10.1002/cyto.990210206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729370PMC
October 1995
2 Citations
3.260 Impact Factor

Evolution of murine alpha 1-proteinase inhibitors: gene amplification and reactive center divergence.

J Mol Evol 1994 Feb;38(2):121-31

Department of Biological Sciences, University of South Carolina, Columbia 29208.

The organization and sequence of genes encoding the alpha 1-proteinase inhibitor (alpha 1PI), a major serine proteinase inhibitor of the mammalian bloodstream, have been compared in several species, including murine rodents (genus Mus). Analysis of gene copy number indicates that amplification of alpha 1PI genes occurred at some time during evolution of the Mus genus, leading to fixation of a family of about three to five genes in several existing species (e.g., M. domesticus and M. saxicola), and only a single gene in others (e.g., M. caroli). A phylogeny for the various mammalian alpha 1PI mRNAs was constructed based upon synonymous substitutions within coding regions. The mRNAs in different murine species diverged from a common ancestor before the formation of the first species lineages of the Mus genus, i.e., about 10-13 million years ago. Thus, alpha 1PI gene amplification must have occurred prior to Mus speciation; gene families were retained in some, but not all, murine species. The reactive center region of the alpha 1PI polypeptide, which determines target protease specificity, has diverged rapidly during evolution of the Mus species, but not during evolution of other mammalian species included in the analysis. It is likely that this accelerated evolution of the reactive center, which has been noted previously for serine proteinase inhibitors, was driven by some sort of a positive Darwinian selection that was exerted in a taxon-specific manner. We suggest that evolution of alpha 1PI genes of murine rodents has been characterized by both modification of gene copy number and rapid reactive center divergence. These processes may have resulted in a broadened repertoire of proteinase inhibitors that was evolutionarily advantageous during Mus speciation.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729375PMC
February 1994
15 Citations
1.680 Impact Factor

Highly conserved upstream regions of the alpha 1-antitrypsin gene in two mouse species govern liver-specific expression by different mechanisms.

Mol Cell Biol 1990 Feb;10(2):760-9

Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.

alpha 1-Antitrypsin (AT), the major elastase inhibitor in mammalian serum, is produced primarily in the liver. We have characterized AT gene structure and expression in the mouse species Mus caroli, which expresses high levels of AT in the kidneys as well as in the liver. Analysis of cDNA and genomic clones showed that the AT gene in M. caroli exhibits high sequence homology (greater than 90%) to the gene in laboratory mice (M. domesticus) throughout the coding and 5'-flanking regions. Despite this extensive sequence conservation, the functional organization of cis-acting regulatory elements governing liver-specific expression is strikingly different between these species. Transient-transfection assays showed that the proximal region of the M. caroli promoter (i.e., between -120 and -2 relative to the transcriptional start site) is 10-fold more active than the analogous region of M. domesticus in driving the expression of an indicator gene in cultured liver cells. The increased activity of the proximal region of the M. caroli AT promoter appears to be the result of one or both of the two base substitutions at positions -46 and -48. The weak proximal promoter in M. domesticus is compensated for by the presence of upstream, liver-specific enhancers between -199 and -520; the analogous region in M. caroli is inactive. Thus, during the course of evolution, the modest 7% sequence divergence that has occurred between the 5'-flanking regions of the AT genes in these two species has generated distinct, yet equally effective, modes of hepatocyte-specific expression.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC360876PMC
February 1990
30 Citations
4.777 Impact Factor

Phorbol ester modulates interleukin 6- and interleukin 1-regulated expression of acute phase plasma proteins in hepatoma cells.

J Biol Chem 1988 Nov;263(33):17390-6

Department of Molecular and Cellular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.

Interleukin 6 (IL 6) and interleukin 1 (IL-1) regulate the expression of acute phase plasma proteins in rat and human hepatoma cells. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), partially mimics the stimulatory effect of IL-6 but reduces that effect of IL-1. TPA and IL-6 act synergistically. These regulatory properties of TPA are also manifested in HepG2 cells transiently transfected with an indicator gene construct carrying the IL-1/IL-6 regulatory enhancer element of the rat alpha 1-acid glycoprotein gene. IL-6 and IL-1 act independently of TPA-inducible kinase C, and of changes in intracellular Ca2+ concentrations. However, prolonged pretreatment of HepG2 cells with TPA results in a drastically reduced cytokine response that is proportional to the loss of cell surface binding activity for the cytokine. These data suggest that hormones activating protein kinase C probably play a contributing role in stimulating the expression of acute phase plasma protein genes but they may be crucial in controlling the responsiveness of liver cells to inflammatory cytokines during subsequent stages of the hepatic acute phase reaction.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729383PMC
November 1988
83 Citations
4.573 Impact Factor

Tissue- and species-specific regulation of murine alpha 1-antitrypsin gene transcription.

J Biol Chem 1988 Oct;263(29):15118-21

Department of Biology, University of South Carolina, Columbia 29208.

Previous studies from our laboratories have shown that the tissue specificity of alpha 1-antitrypsin (AT) expression differs among closely related mouse species. In laboratory mice (Mus domesticus), AT mRNA is found almost exclusively in the liver. In the wild-derived species Mus caroli, the mRNA is expressed not only in the liver but also in the kidney, where it is regulated by androgens during post-natal development. We presently show that the tissue specificity, the species specificity, and the developmental regulation of AT mRNA levels correlate with the transcription rate of the AT gene, as measured by nuclear run-on assays. During the course of these experiments, we found that some AT-specific probes are complementary to constitutively synthesized RNAs that do not accumulate and that are unrelated to functional AT mRNA expression. These RNAs, which result from both sense and anti-sense transcription, may derive from aberrant initiation events within certain regions of the AT gene.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729444PMC
October 1988
10 Citations
4.573 Impact Factor

Developmental expression, cellular localization, and testosterone regulation of alpha 1-antitrypsin in Mus caroli kidney.

J Biol Chem 1987 Sep;262(26):12641-6


 


?1-Antitrypsin (?1-protease inhibitor), an essential plasma protein, is synthesized predominantly in the liver of all mammals. We have previously shown that Mus caroli, a Southeast Asian mouse species is exceptional in that it expresses abundantly ?1-antitrypsin mRNA and polypeptide, in the kidney as well as the liver (Berger, F. G., and Baumann, H. (1985) J. Biol. Chem. 260, 1160–1165) providing a unique model for examination of the evolution of genetic determinants of tissue-specific gene expression. In the present paper, we have further characterized ?1-antitrypsin expression in M. caroli. The extrahepatic expression of ?1-antitrypsin is limited to the kidney, specifically within a subset of the proximal tubule cells. The developmental pattern of ?1-antitrypsin mRNA expression in the kidney differs from that in the liver. In the kidney, ?1-antitrypsin mRNA is present at only 2–4% adult level at birth and increases very rapidly to adult level during puberty between 26 and 36 days of age. There are no significant changes in liver ?1-antitrypsin mRNA levels during this period. Testosterone, while having only modest affects on ?1-antitrypsin mRNA accumulation in the adult kidney, causes a 20-fold induction of the mRNA in the pre-pubertal kidney. This suggests that the increase in ?1-antitrypsin mRNA expression during puberty is testosterone mediated. Southern blot analyses of Mus domesticus and M. caroli genomic DNA and a cloned M. caroli ?1-antitrypsin genomic sequence, indicate that a single ?1-antitrypsin gene exists in M. caroli, whereas multiple copies exist in M. domesticus. These data show that the alteration in tissue specificity of ?1-antitrypsin mRNA accumulation that has occurred during Mus evolution is associated with distinctive developmental and hormonally regulated expression patterns.


View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729446PMC
September 1987
16 Citations
4.573 Impact Factor

Mouse alpha 1-protease inhibitor is not an acute phase reactant.

Arch Biochem Biophys 1986 Apr;246(1):488-93

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.

View Article

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4743027PMC
April 1986
21 Citations
3.017 Impact Factor

Top co-authors

Stephen G Grant
Stephen G Grant

Graduate School of Public Health

12
Crystal M Kelly
Crystal M Kelly

University of Pittsburgh School of Medicine

4
Jennifer M Johnson
Jennifer M Johnson

University of Oklahoma Health Sciences Center

4
Victor G Vogel
Victor G Vogel

University of Pittsburgh School of Medicine

3
Tiffany D Miles
Tiffany D Miles

University of Pittsburgh Cancer Institute

3
Wendy S Rubinstein
Wendy S Rubinstein

National Center for Biotechnology Information

3
Joseph L Kelley
Joseph L Kelley

University of Pittsburgh School of Medicine

2
Michael J Forlenza
Michael J Forlenza

Simon Fraser University

2