Abstract
Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems.
Highlights
Apoptosis is a common form of programmed cell death occurring in metazoans that leads to removal of cells in the organism while avoiding the induction of inflammation [1,2]
The resulting yeast strain, DSI (BAK), displayed significant killer protein expression following thiamine removal and died with great efficiency after three rounds of replica plating on thiamine-free yeast agar plates
We had demonstrated quantitative cell death in the DSI strain in liquid culture 24 hours after BAK induction [15]. We used another S. pombe yeast strain with inducible killer protein expression, HC4, in which the C. elegans homologue of APAF-1, CED-4 [37], had been cloned into the pRIP45 vector and stably integrated
Summary
Apoptosis is a common form of programmed cell death occurring in metazoans that leads to removal of cells in the organism while avoiding the induction of inflammation [1,2]. The extrinsic pathway is physiologically triggered by ligands of the death receptor family, which leads to receptor clustering, assembly of the cytoplasmic receptor complex DISC (death inducing signaling complex) and activation of initiator CASPASE-8 and CASPASE-10 within the DISC platform [3]. While apoptosis functions to coordinate the elimination of excess, hazardous or damaged cells under normal physiological conditions [2,5], alterations in the regulatory mechanisms of cell death/survival contribute to many human pathological conditions, including cancer and neurodegenerative diseases, highlighting the importance of maintaining the tight regulation of the apoptotic machinery [6]. Tumor cells are usually more sensitive to external apoptotic stimuli compared with their non-transformed counterparts, thereby indicating that despite acquisition of apoptosis-inhibiting mutations, they are pushed to the edge of survival by environmental (and proliferative) stresses [11,12]. The developing resistant tumor metastases represent a severe clinical problem and significantly contribute to the cancer-related death rate [14]
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