Dianne C Daniel, PhD
- ADJUNCT ASSISTANT PROFESSOR | Pathology
Eastern Virginia Medical School, Department of Microbiology and Molecular Cell Biology, Lewis Hall, Room 3152, 700 West Olney Road, Norfolk, VA 23507-1696
Applications accepted from committed researchers.
Our research is an exploration of molecular mechanisms underlying eukaryotic DNA replication and how these mechanisms are targeted during cancer progression. Discovery at this level is aimed at translation into clinical studies for the prevention and treatment of cancer and the bioengineering of novel protein applications. We use both cell culture and human pathology specimens to perform studies that compliment each other. These studies involve various cellular, molecular, biochemical and biophysical approaches.
Molecular, cellular and biochemical analysis of MCM8 function
The role of the minichromosome maintenance (MCM) proteins in initiation of DNA replication has been extensively studied, and the MCM2-7 toroid is proposed to be a replicative helicase unwinding DNA ahead of the replication fork during S phase. Additional functions are currently being ascribed to these family members. In 2003, we discovered the gene for MCM8, a new family member that is not found in yeast, but which has seemly evolved to adapt to the more complex nuclear structure of higher eukaryotes. We, and others, have shown the MCM8 gene to be either mutated or variantly spliced in a choriocarcinoma and virally interrupted in a hepatocarcinoma, respectively. We have reported that MCM8 is reduced in colon cancer. We are performing in vitro biochemical characterization of MCM8 activity relating to DNA replication and repair. Through such techniques as immunoprecipitation, electron microscopy, confocal microscopy and mass spectrometry, we are also identifying functional interactions of MCM8 with components of the replisome and with cellular regulators. We verify the simultaneous presence of two proteins at genomic sequences representing a characterized origin of replication using double chromatin immunoprecipitation (ChIP) procedure. We are applying information from the various analyses to studies of MCM8 interplay with regulatory pathways within the cell using mouse and human models.
We have collected ovarian cancer specimens and prepared breast organoids to model studies complementary to our cell culture studies. When possible, we match normal and tumor specimens from the same patient. Using the techniques of immunohistochemistry, dual immunofluorescence microscopy, laser capture microdissection combined with real-time RT- PCR and expression arrays, we are examining MCM8 expression at the level of both RNA and protein. These studies complement knockdown and mutational studies derived from our cell culture models.
Kinoshita Y, Johnson EM, Gordon RE, Negri-Bell H, Evans MT, Coolbaugh J, Rosario-Peralta Y, Samet J, Slusser E, Birkenbach MP, Daniel DC. Colocalization of MCM8 and MCM7 With Proteins Involved in Distinct Aspects of DNA Replication. Microsc Res Tech 2008; 71(4): 288-297.
Daniel DC, Kinoshita Y, Khan MA, Valle LD, Khalili K, Rappaport J, Johnson EM. Internalization of exogenous human immunodeficiency virus-1 protein, Tat, by KG-1 oligodendroglioma cells followed by stimulation of DNA replication initiated at the JC virus origin. DNA Cell Biol 2004 Dec; 23(12): 858-867.
Johnson EM, Kinoshita Y, Daniel DC. A new member of the MCM protein family encoded by the human MCM8 gene, located contrapodal to GCD10 at chromosome band 20p12.3-13. Nucleic Acids Res 2003; 31(11): 2915-2925.
Daniel DC. Comment Article. BRCA1 and BRCA2 proteins in cell function. In: Wolvey W, editor. Breast Cancer
Comment Article. BRCA1 and BRCA2 proteins in cell function. London, Current Medical Literature Ltd.; 2003. pp1-9.
Khalili K, Del Valle L, Muralidharan V, Gault WJ, Darbinian N, Otte J, Meier E, Johnson EM, Daniel DC, Kinoshita Y, Amini S, Gordon J. Pura is essential for postnatal brain development and developmentally-coupled cellular proliferation as revealed by genetic inactivation in the mouse. Mol Cell Biol 2003; 23(19): 6857-6875.
Daniel DC. Editorial. Breast cancer: cell biology, clinical parameters and imaging. Microsc Res Tech 2002; 59(1): 1-2.
Daniel DC. Highlight: BRCA1 and BRCA2 proteins in breast cancer. Microsc Res Tech 2002; 59(1): 68-83.
Daniel DC, Wortman MJ, Schiller RJ, Liu H, Gan L, Mellon JS, Chang CF, Gallia GL, Rappaport J, Khalili K, Johnson EM. Coordinate effects of human immunodeficiency virus type-1 protein, Tat, and cellular protein, Pur-alpha, on DNA replication initiated at the JC virus origin. J Gen Virol 2001; 82(Pt 7): 1543-1553.
Daniel DC. Dual immunofluorescence labeling with cell-specific markers localizes BRCA1 in both basal and luminal epithelial cells in primary outgrowth from noncancerous mammary ductal and alveolar preparations. Cell and Tissue Res 1999; 298(3): 481-487.
Daniel DC, Weston A. Benzo(a)pyrene enhances levels of ErbB2 in normal mammary epithelial cells in organoid culture. Cancer: Genetics and the Environment, Ann NY Acad Sci 1997; 833: 169.
Berberich S, Trivedi A, Daniel DC, Johnson EM, Leffak M. In Vitro Replication of Plasmids Containing Human c-myc DNA. J Mol Biol 1995; 245(2): 92-109.