The Research Laboratory of Debananda Pati, PhD
The general area of research in the Pati laboratory includes chromosomal cohesion and separation and their role in the development of aneuploidy, tumorigenesis and regulation of apoptotic cell death. Aneuploidy (abnormal chromosome number) has been described for over a century and is a feature of many tumor cells. Although there have been many proposed hypotheses, there is no agreement as to why aneuploidy is so highly prevalent in cancer cells and if it contributes to tumor progression. The mechanisms of aneuploidy therefore remain a fundamental unresolved issue in cancer biology. The broad goal of our laboratory is to understand the mechanisms of aneuploidy and to apply this knowledge to cancer therapy.
Genetic instability often manifests as a loss or gain of whole chromosomes (aneuploidy) and occurs in many cancer types. Aneuploidy can result from the loss of coordination between cell division and programmed cell death (apoptosis), which are both critical for proper cellular function. Our laboratory is interested in identifying proteins that coordinate aneuploidy, cell division, and apoptosis in cancer tissues in an effort to understand the regulatory network that assures accuracy of chromosomal separation in dividing cells. Premature or erroneous separation of sister chromatids or homologous chromosomes can result in birth defects or inherited genetic diseases, including cancer. Our research is aimed at elucidating the molecular mechanism of chromosomal instability through analysis of the factors that mediate sister chromatid cohesion and separation during mitosis.
Both estrogen and progesterone are critical hormones in mammary development in rodents and humans, and there is compelling evidence that both of these steroid hormones promote tumorigenesis in humans. Recent studies in mouse models deficient in the tumor suppressor gene p53 indicate that progesterone can significantly enhance chromosomal instability in p53 null mammary cells as determined by aneuploidy status. Understanding the molecular controls of chromosomal instability, cell death in relation to chromosomal segregation, and the effect of hormones will provide important information on factors that underlie both the carcinogenic process itself and the response to treatment. Ongoing projects in our laboratory aim to dissect the molecular mechanisms of aneuploidy by evaluating the effect of steroid hormones on the mitotic machinery, particularly on sister chromatid cohesion and separation and mitotic checkpoint signaling. Drawing a relation between steroid hormone signaling and mitotic checkpoint control and sister chromatid cohesion is novel and will lead to greater understanding of the link between steroid-induced chromosomal instability and aneuploidy. Understanding the underlying mechanisms of steroid-induced aneuploidy may also reveal therapeutic targets in various cancers.
Current therapies for hematopoietic malignancies are based on reducing cancer cell proliferation and promoting programmed cell death (apoptosis). Much of the current effort in cancer biology is concentrated on studying either the cell cycle or the apoptotic pathways individually, and relatively little is known about the coregulation of these two vital processes. Understanding the common pathways that regulate both cell proliferation and apoptosis would provide a new paradigm for identifying novel therapies for hematologic cancers. We propose that the processes of mitotic segregation and apoptosis are mechanistically linked and that proteins important for sister chromatid cohesion and separation play a role in regulating normal apoptotic processes. Deregulation of this joint process can lead to the formation and progression of hematologic cancers, including leukemia and lymphoma. During DNA replication, a group of conserved proteins termed cohesins forms a complex that holds the two sister chromatids together to ensure accurate chromosomal segregation during the normal mitotic cell cycle. Human Rad21 protein along with at least three other subunits (SCC3, SMC1, and SMC3) are components of such a sister chromatid cohesin complex. Proteolytic cleavage of this complex by the protease Separase at the metaphase-to-anaphase transition triggers the separation of sister chromatids. Unexpectedly, data from our lab and another indicate that Rad21 is cleaved early in apoptosis in a number of leukemic T- and B- cell lines, including Molt4, Jurkat and Reh by a caspase-like molecule. After cleavage of Rad21, carboxyl-terminal (C-terminal) Rad21 is translocated to the cytoplasm and promotes apoptosis in leukemia cell lines. Cleavage of cohesin Rad21 is carried out by a Separase in mitosis and by an unidentified caspase/caspase-like molecule in apoptosis at different sites in the protein. Both of the proteases belong to the distantly related CD clan protease family, suggesting an evolutionarily conserved mechanism shared by the mitotic and apoptotic machinery. It is therefore possible that chromosomal segregation proteins such as Rad21 and Separase may serve as links between the two key cellular processes of mitosis and apoptosis. Deregulation of this pathway may lead to treatment-resistant disease. The central hypothesis that mitotic segregation and apoptosis are linked processes, is currently being tested by studying the function of cohesin Rad21.
Our laboratory has also been interested in the role of ubiquitin-mediated proteolysis and its role in the genesis and development of cancer. We study a number of proteins in this pathway including a ubiquitin-related protein called ISG15 (aka Interferon Stimulated Gene product, 15kDa) and two key enzymes of ubiquitination, human Rad6B and mammalian Cdc34 proteins. We use various approaches including a two-hybrid screen to identify proteins that interact with human ISG15, Cdc34 and Rad6B, and developing a mouse model by targeted disruption of the murine ISG15 gene via homologous recombination. The goal of these studies is to evaluate their impact on hematopoiesis, and to characterize the expression of ISG15 and Cdc34 and its interacting proteins in leukemia samples and tissues.
Dr. Pati’s laboratory is part of the Cancer Genetics and Genomics Program and the Solid Tumor Program’s sarcoma research laboratories.
My laboratory is performing research in two main areas: 1) investigation of the molecular basis of aneuploidy in breast cancer models, and 2) mitotic regulation of apoptosis in pediatric leukemia. In an effort to understand the regulatory network that assures accuracy of chromosomal separation in dividing cells, we have identified a set of chromosomal segregation proteins that coordinate aneuploidy, cell division, and apoptosis in cancer tissues. Our research is aimed at elucidating the molecular mechanism of chromosomal instability through analysis of the factors that mediate sister chromatid cohesion and separation during mitosis. My lab has recently proposed a new handcuff model for the chromosomal cohesin complex, and has also demonstrated that overexpression of Separase, the cohesin protease that cleaves cohesin subunit Rad21 during mitosis, causes aneuploidy and tumorigenesis. The current focuses of my lab are: 1) the structural and functional analysis of the cohesin complex, 2) the regulation of Separase expression and aneuploidy by p53 and steroid hormones using Separase and securin transgenic mice models, 3) designing and screening small molecular inhibitors against Separase enzyme active site and testing their efficacy for tumor regression using our recently developed Separase inducible mouse mammary tumor model, and 4) identification and characterization of a novel nuclear protease that cleaves cohesin Rad21 for apoptosis induction.
Sr. Staff Scientist
Nenggang Zhang, PhD
Maintaining genomic stability is the most important task for cells. In addition to genetic mutations, the majority of cancer cells lose their genomic integrity due to gains or losses of chromosomes, gross chromosomal rearrangements, and amplification or deletion of genetic material. I am interested in identifying the molecular mechanisms that mediate the genomic integrity in normal cells and genetic instability in cancer cells. My research utilizes various methods including genomic, biochemical, molecular structure, and cellular biological approaches. Currently I focus on the mechanisms of sister chromatid cohesion and segregation.
Anil Panigrahi, PhD
The cohesin component Rad21 is cleaved during apoptosis, and the resultant C-terminal fragment translocates to the cytoplasm to accelerate the apoptotic response. I am working on purifying and identifying the proteinase that cleaves Rad21 during apoptosis. I am also working on protein-interactors of Rad21 to gain insight into the wider picture of cohesin function. I am interested in cohesin biology, eukaryotic epigenetics and gene regulation.
Elizabeth McCallum, PhD
(Supported by the Ladies Auxiliary to the Veterans of Foreign Wars of the United States)
Towards a new approach for breast cancer therapy our lab has recently proposed the novel concept of disrupting the normal association of the cohesin complex that cells require for successful cell division. I will use a unique molecular screening process to identify small protein fragments that block the cohesin complex from forming, thus leading to abnormal separation of genetic material in the rapidly growing cancer cells and triggering their self destruction.
I am interested in the structural study of the cohesin complex with EM and crystallography methods. I am currently working on the structure of the cohesin subunit SA2 as well as the interaction between Rad21 and SA2.
Lab Manager and Research Assistant
Gouqing (Mary) Ge, BS
As the manager of the Pati Lab, I look after the day-to-day functioning of the laboratory. I am also actively participating on many ongoing projects including Separase mutant mouse models. I am particularly interested in long term culture of primary and human embryonic stem cells.
The University of Texas at El Paso
Embedding of Mouse Embryo
Detecting Ubiquitinated Protein on a Membrane
Genomic DNA from mouse tail
Pati lab SDS-PAGE/Western Protocol
Southern blot Protocol
Tissue Processing for Histology
Silver Staining of Protein Gels
Targeting solid tumors and breast cancer