Biomedical subjects

Ameeta Kelekar

Publications and source records attributed to Ameeta Kelekar.

4 recordsLinked to original sources

PubMed2005

Multi-probe RPA template sets to study RNA modulation and transcriptional control of BH3-only members of the Bcl-2 family.

The Bcl-2 family of apoptotic regulators comprises both pro- and anti-apoptotic proteins. Pro-apoptotic BH3-only members of the Bcl-2 family are subject to stringent control in normal proliferating cells, but can be activated by a variety of mechanisms in response to diverse apoptotic signals. To facilitate the simultaneous identification of BH3-only targets that are regulated at the RNA level, we have designed and assembled multi-probe ribonuclease protection assay templates and demonstrated their ability to detect multiple BH3-only transcripts in a variety of human cell lines and primary tissues. Following this, we have determined the modulation of BH3-only transcripts to a variety of apoptotic stimuli, in selected primary and transformed cell lines. Early experiments, showing that a given cell type upregulates a unique subset of BH3-only RNAs in response to different death-inducing stimuli, indicate that the multi-probe BH3-only RPA template sets could serve as useful diagnostic and analytical tools in cancer research.

Apoptosis↗

PubMed2005

Autophagy.

Autophagy is a major intracellular pathway for the degradation and recycling of long-lived proteins and cytoplasmic organelles. Like apoptotic programmed cell death, autophagy is an essential part of growth regulation and maintenance of homeostasis in multicellular organisms. Autophagic vacuole formation is also activated as an adaptive response to a variety of extracellular and intracellular stimuli, including nutrient deprivation, hormonal or therapeutic treatment, bacterial infection, aggregated and misfolded proteins and damaged organelles. Mediators of class I and class III PI3 kinase signaling pathways and trimeric G proteins play major roles in regulating autophagosome formation during the stress response. Defective autophagy is the underlying cause of a number of pathological conditions, including vacuolar myopathies, neurodegenerative diseases, liver disease, and some forms of cancer. This chapter provides an overview of the morphology and molecular basis of autophagosome formation and offers a glimpse into the role of autophagy in normal growth and development, while discussing the pathological implications of its deregulation.

Animals↗

PubMed2004

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L).

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.

Annexin A5↗

PubMed2003

Retroviral immunotoxin gene therapy of leukemia in mice using leukemia-specific T cells transduced with an interleukin-3/Bax fusion protein gene.

In past studies, we showed that T cells transduced with retroviral diphtheria immunotoxin (IT) target genes could serve as vehicles for delivering IT to tumors in vivo. We took advantage of the observation that antigen-specific T cells are able to penetrate tumors to design an approach delivering combined cellular and humoral therapy directly to the tumor site. To improve tumor specificity, we selected interleukin (IL)-3 as a ligand because its receptor is selectively overexpressed on myeloid leukemia progenitors. Because Bcl-2 family proteins show structural similarity to diphtheria toxin (DT), we constructed a unique retroviral IT using Bax, a proapoptotic member of the Bcl-2 family, in place of DT. Bax was chosen because several studies showed that its transduction induces lethal apoptosis in different cancers. The retroviral construct for gene therapy included IL-3 positioned downstream of its 80 amino acid leader, and permitted cotranslational protein synthesis of hybrid IL-3/human Bax fusion protein. Other vectors were constructed with IL-3 fused to DT or Pseudomonas exotoxin. Retroviral vectors were used to transiently transduce C8, a CD4(+) T cell clone that specifically recognized FBL-3, a lethal myeloid leukemia. Supernatants collected from transduced cells showed proapoptotic activity and selectively inhibited FBL-3 cells in vitro. Intraperitoneal injection of transduced but not nontransduced C8 into mice with subcutaneous tumors or systemic cancer significantly inhibited tumor growth. These results indicate that retroviral IT made with IL-3 and various toxic proteins may be useful in patients with acute myelogenous leukemia (AML). Furthermore, the Bax construct may be particularly useful as a nonimmunogenic substitute for bacterial toxins in retIT.

Animals↗