Functional analysis of s6k1 regulation of apoptosis control
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Lindsay M. Webb, Catherine A. Gallo, David R. Plas. Department of Cancer Cell Biology, University of Cincinnati. Abstract

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Functional analysis of S6K1 regulation of apoptosis control

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Functional analysis of s6k1 regulation of apoptosis control

Lindsay M. Webb, Catherine A. Gallo, David R. Plas

Department of Cancer Cell Biology, University of Cincinnati

Abstract

Increased glucose-dependence characterizes the altered metabolism of tumor cells. S6K1 is a protein kinase that promotes glycolysis and glucose-dependent survival in leukemia cells. In cells expressing activated S6K1 or the vector control, we tested cell survival by treatment with an S6K1 inhibitor. S6K1 was required for survival, but activated S6K1 was not sufficient for survival. The data support the potential efficacy of S6K1 inhibition in cancer chemotherapy.

Introduction

PTEN deficiency activates protein kinase, S6K1, which activates glycolysis and glucose-dependent survival.ΔnΔc S6K1 is a constitutively active S6K1 protein kinase. Therefore, cells containing the ΔnΔc S6K1 construct should maintain a higher viability under apoptotic conditions. AD80, an S6K1 inhibitor, should promote apoptosis.

Results

Figure 1: Cloning of ΔnΔc S6K1 into MIT vector.

A.

B Map of ΔnΔc S6K1 construct C. Digest verification

Figure 4: Role of S6K1 in survival control

A. B.

Figure 4: S6K1 is necessary, but not sufficient for survival. A. Structure of AD80, an S6K1 inhibitor. B. Cells were starved of IL3, a growth factor, to test survival control. PTEN deficient cells survive better than control or FL5.12s in vehicle treated cells. ΔnΔc S6K1 did not improve survival in any cell type. The S6K1 inhibitor, AD80, prevented the survival of PTEN deficient cells.

Conclusion

Activated S6K1 is not sufficient for survival. S6K1 inhibition shows that S6K1 is required for survival.

Insufficient:

Required:

Acknowledgements

University Honors Program

SURF Program

Plas Research Group, University of Cincinnati

Shokat Research Group, University of California, San Francisco

Figure 2: Transfection and transduction of ΔnΔc S6K1 into three FL5.12 cell lines

A

B

C

Functional analysis of S6K1 regulation of apoptosis control

sort

97%

27%

Figure 2: Immortalized, hematopoietic FL5.12 cell lines were transduced and sorted. A. Parental, Control, PTEN deficient cell lines. B. Model of retroviral production and target cell transduction. C. Transduced cells were stained for Thy1-1 (a transduction marker) then sorted to >95% Thy1-1+.

Figure 3: ΔnΔc S6K1 Validation

A Testing Expression B Testing Substrates

VecPTEN-FL5.12

VecPTEN-FL5.12

ΔnΔc - + - + - +

Myc tag

PTEN

ΔnΔc - + - + - +

S6

pS6

NcoI

uncut

ladder

Figure 3: ΔnΔc S6K1 construct was validated by Western blot. A. Myc tag indicates the expression of the ΔnΔc construct in Vector, PTEN-, and FL5.12. B. S6 is increasingly phosphorylated in PTEN-deficient cells. There is little significant difference of ΔnΔc S6K1 on phosphorylation of S6 in all three cell types.

Figure 1: Cloned ΔnΔc S6K1 into retroviral vector, MIT. A. Model of S6K1. ΔnΔc S6K1 has previously been shown to be an activated kinase.B. Map of ΔnΔc S6K1 MIT. B. Verification digest by NcoI produced 6.0 kb and 1.2 kb fragments.


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