Browsing by Subject "Bcl-2"

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Cancer cells, however, can express constitutively active oncogenic kinases such as BCR-Abl that promote these processes independent of extrinsic growth factors. When cells are deprived sufficient growth signals or when oncogenic kinases are inhibited, glucose metabolism decreases and cells activate the self-digestive process of autophagy, which clears damaged organelles and provides degradation products as an alternate fuel to support mitochondrial metabolism. Importantly, loss of growth signals can also lead to apoptosis mediated through Bcl-2 family proteins, and Bcl-2 has been reported to interfere with autophagy, potentially disrupting a key nutrient source just as glucose uptake becomes limiting. Since autophagy may support survival or lead to death depending on context, the role of this pathway in apoptosis-competent growth factor deprived cells remains unclear.

In this thesis, I examine the interactions of autophagy with Bcl-2 family proteins and apoptosis upon inhibition of growth signals in hematopoietic cells. In contrast to other studies, I found autophagy was rapidly induced in growth factor deprived cells regardless of Bcl-2 or Bcl-xL expression, and this led to increased production of fatty acids and amino acids for metabolism. While these data suggested autophagy may play a key role to support metabolism of growth factor deprived cells, provision of exogenous pyruvate or lipids as alternate fuel had little affect on cell survival. Instead, I found that autophagy modulated cell stress pathways and Bcl-2 family protein expression in a context specific fashion to impact cell fate.

My results show that autophagy's effect on cell survival is dependent on its level of induction within a cell. I observed that partial suppression of autophagy protects cells from stress and induction of pro-apoptotic Bcl-2 family expression, while complete inhibition of autophagy enhances stress and is pro-apoptotic. In experiments using shRNAi to partially suppress autophagy, I found increased survival upon growth factor deprivation in several different types of cells expressing anti-apoptotic Bcl-2 or Bcl-xL, indicating that autophagy promoted cell death in these instances. Cell death was not autophagic, but apoptotic, and relied on direct Chop-dependent transcriptional induction of the pro-apoptotic Bcl-2 family protein Bim. In contrast, complete acute disruption of autophagy through conditional Cre-mediated excision of the autophagy-essential gene Atg3 led to p53 phosphorylation, upregulation of p21 and the pro-apoptotic Bcl-2 family protein Puma, and rapid cell death of cells the presence or absence of growth factor. Importantly, transformed BCR-Abl-expressing cells had low basal levels of autophagy but were highly dependent on this process. Deletion of Atg3 or treatment with chemical autophagy inhibitors led to rapid apoptosis, and BCR-Abl expressing cells were unable to form leukemia in mice in without autophagy. Together, my data demonstrate a dual role for autophagy in cell survival or cell death and suggest that the level of autophagy in a cell is critical in determining its role in apoptosis and cell fate. Ultimately, these results may help to determine future approaches to modulate autophagy in cancer therapy.

Hematopoiesis is the continual process of blood cell generation that primarily occurs in the bone marrow of adult animals. Hematologic neoplasms can also occur in the bone marrow and often result from dysregulation of signal transduction pathways. One example is the activation of the Ras oncogene, which has been linked to a variety of different cancers, including hematologic neoplasms. Ras is located proximal to the cell membrane and can activate many downstream effector pathways, thus it is difficult to determine which downstream pathway is mediating oncogenic Ras function. My thesis work focused on the effect of inappropriate activation of MEK/ERK, a downstream Ras effector pathway, in the hematopoietic system.

Using a retroviral transduction system, we expressed a constitutively active form of MEK1 in hematopoietic stem cells (HSCs). Mice transplanted with HSCs expressing active MEK developed a lethal myelodysplastic syndrome/myeloproliferative disease (MDS/MPN) characterized by the expansion of granulocytes/macrophages (GM) at the expense of lymphoid cell development. Transplantation of active MEK-induced MDS/MPNs into naïve mice did not result in further disease, suggesting that the MDS/MPN is not a frank leukemia.

Bcl-2 is an anti-apoptotic molecule that has been shown to play a role in leukemia development and maintenance. Coupling expression of active MEK and Bcl-2 resulted in MDS/MPNs that were phenotypically identical and had very similar disease onset compared to active MEK-induced MDS/MPNs. However, transplantation of Bcl-2/active MEK-induced MDS/MPNs did not result in a myeloid disease; rather, it resulted in the development of T-acute lymphoblastic leukemia (T-ALL) that was marked by activated Notch signaling.

These results led us to conclude that activation of MEK/ERK was sufficient to cause a pre-leukemic myeloid disease; however, additional oncogenic factors, such as Bcl-2 and Notch, were necessary for frank leukemia development. Moreover, additional oncogenic factors can alter the disease phenotype and disease course. Future analysis of the interplay between oncogenic factors will help shed light on disease development and aid in the development of more effective cancer treatments.

Throughout their development in the thymus and during their maintenance and the immunological response in the periphery, T cells rely on the regulation of classical apoptotic pathways to promote cell survival or death. Several proteins of the Bcl-2 family have been shown to be critical in thymocyte and T cell survival and consequently, in T cell function. Among these proteins, the antiapoptotic proteins Bcl-2 and Mcl-1 are critical for promoting T cell survival at multiple stages of the T cell "life cycle." While these proteins have been reported to interact with several of the proapoptotic members of the Bcl-2 family, the specific interactions by which Mcl-1 in particular promotes T cell survival in vivo were not well understood. Further, how different stimuli (for example, cytokine signaling and T cell activation) modulate the specific functions of Mcl-1 had also not been thoroughly explored.

We utilized mouse models to dissect the roles of Mcl-1 at multiple stages of T cell development and function. We utilized conditional knockout and double knockout strategies to build genetic pathways for Mcl-1 activity during thymocyte development and in peripheral T cells under a variety of conditions. In the thymus, the major role of Mcl-1 is to inhibit the activity of proapoptotic Bak because the loss of Bak, but not the loss of Bax or Bim, rescued the survival of Mcl-1-deficient thymocytes at both the double negative and single positive stages. Further, we concluded that this role is not shared with Bcl-2 because overexpression of Bcl-2 did not rescue DN or SP survival.

In peripheral T cells, the loss of Bak rescued T cell survival in the presence of IL 7, but not during conditions of cytokine withdrawal. Interestingly, the overexpression of Bcl-2 or the loss of Bim partially rescued the survival of T cells during cytokine withdrawal, indicating that Mcl-1 has dual roles in T cells: cytokine-dependent and cytokine independent. Additionally, we found that cytokines of the common gamma chain family have different effects on the activity of Mcl-1 due to the differential regulation of other proteins of the Bcl-2 family, most notably Bim.

Finally, we utilized a Bcl-2 reporter mouse model to examine the role of Bcl-2 in the establishment of CD8+ T cell memory to infection. Although it is known that Bcl-2 is dynamically regulated in response to activation, the importance of this regulation in the establishment of T cell memory is not yet clear. We show that a subset of effector T cells within a previously defined memory precursor population retained high Bcl-2 expression at the peak of the immune response. Using adoptive transfer of sorted effector T cells, we provide preliminary evidence that the cells with memory potential lie within a strict range of Bcl-2 expression. These studies indicate that the regulation of Bcl 2 is likely critical in establishing T cell memory and provide a platform for the future study of the factors that influence T cell memory.