LIFE09200604002 Lalit Sehgal - Homi Bhabha National Institute

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to WT mice. This was confirmed by immunohistochemical analysis using antibodies to CD3. To confirm whether the splenomegaly was due to an increase in lymphoid cells in the spleen, the spleens of WT or 14-3-3ε knockdown mice were stained with antibodies to CD3 (T cell marker). The spleen sections of the 14-3-3ε knockdown mice showed an increased presence of CD3 and CD45 positive lymphocytes as compared to WT mice. The spleen cells were purified from mice and an immunophenotyping experiment was performed using antibodies that recognize the CD3, CD4, CD8 and B220 antigens. It was observed that there is increase in the CD4+ population in the cells purified from the spleens of the knockdown mice as compared to the vector control. We also found that the CD4-/CD8- double negative T cell population is increased in cells purified from the spleen of knockdown mice. Interestingly; multiple reports have suggested that 14-3-3ε levels are reduced in adult T-cell leukemias. In addition, we have also observed that there is an increase in the number of CD3+ positive cells in the lungs of the knockdown mice as compared to the vector controls. It has been reported that a downstream effector of T cell activation, SLP-76, is important to maintain T cells in activated state (164). An in silico analysis of SLP-76 using Motif Scan revealed the presence of a 14-3-3 binding motif in SLP-76 and the hematopoietic protein kinase 1 phosphorylates SLP-76 at Serine residue 376 to generate 14-3-3 binding motif (79). To determine if 14-3-3ε can form a complex with SLP76 in the presence of HPK1, FLAG-SLP76, and HA 14-3-3ε were transfected into HEK 293 cells in the presence or absence of HA-HPK1. It was observed that SLP76 formed a complex with 24
14-3-3ε only in the presence of HPK1. This result suggested that 14-3-3ε is negatively regulating T cell activation by binding to and inhibiting SLP76 function. We also observed swollen lymph nodes in some of the 14-3-3ε knockdown mice. To determine if the swollen lymph nodes were due to the presence of transformed cells, pieces of the lymph node were transplanted on to the backs of SCID mice. The lymph nodes were capable of forming a transplantable tumor. A PET analysis using 5FDG demonstrated that the tumor is vascularized and capable of initiating the formation of blood vessels. These results suggest that 14-3-3ε downregulation may lead to neoplastic transformation. To determine whether 14-3-3ε can inhibit tumor formation, stable cell clones lacking 14-3-3ε generated in NIH3T3 cells were tested for their ability to form colonies in soft agar as compared to the vector control. The 14-3-3ε knockdown clone E17, formed larger and more colonies in soft agar as compared to the vector control. The migration ability of cells lacking 14-3-3ε increased as determined by wound healing assay. In addition we determined whether the NIH3T3 derived 14-3-3ε knockdown clone can form tumors in SCID mice. Five out of six injected mice showed the presence of tumor as compared to none for the vector control. These results suggest that loss of 14-3- 3ε can lead to neoplastic transformation. The 14-3-3ε knockdown mice show patchy hair loss. To determine whether the organization of the epidermis is altered in the 14-3-3ε knockdown clones, skin sections from WT or mutant mice were stained with hematoxylin and eosin. We observed epidermal stratification is altered in the skin of 14-3-3ε knockdown mice as compared to the wild type mice. Hair follicles and the hair shaft develop normally in the WT mice however in the 14-3-3ε knockdown mice the hair shaft did not develop and hair follicles 25
Page 1 and 2: Generation of knockdown mice that l
Page 3 and 4: STATEMENT BY AUTHOR This dissertati
Page 5 and 6: CERTIFICATE I certify that the thes
Page 7 and 8: I am thankful to Mr Uday Dandekar f
Page 9 and 10: 3.16 Isolation of Genomic DNA (gDNA
Page 11 and 12: A synopsis of thesis to be submitte
Page 13 and 14: esults in loss of 14-3-3 binding an
Page 15 and 16: and NheI (NEB) to remove tetO-CMV,
Page 17 and 18: Hanging drop and wound healing assa
Page 19 and 20: 360). To generate transgenic animal
Page 21 and 22: asement of the seminiferous tubules
Page 23: normal female, pups were screened f
Page 27 and 28: proteins are expressed in testis (3
Page 29 and 30: 5. Lalit Sehgal, Srikanth B., Khyat
Page 31 and 32: Presented a poster at 33rd All Indi
Page 33 and 34: shRNA siRNA TBS-T U.V WT C IF DSG D
Page 35 and 36: List of Figures: Page No 1. Figure
Page 37 and 38: 37. Figure 4.3.20: 14-3-3γ interac
Page 39 and 40: CHAPTER 1 INTRODUCTION 39
Page 41 and 42: 1.1.1 Cyclin Cdk complexes Eukaryot
Page 43 and 44: 1.1.1.c Cyclin A cdk complexes The
Page 45 and 46: of which are associated with acquis
Page 47 and 48: 1.2.2 The G1/S DNA Damage checkpoin
Page 49 and 50: complexes. DNA damage induced degra
Page 51 and 52: cytoplasmic accumulation of cdc25 p
Page 53 and 54: 14-3-3 binding site. 14-3-3 binding
Page 55 and 56: G2 arrest and induced premature chr
Page 57 and 58: emoval of S216 phosphorylation as i
Page 59 and 60: embryogenesis, a loss of cdc25A can
Page 61 and 62: dimer was essential for Raf activat
Page 63 and 64: of enhanced apoptosis of peripheral
Page 65 and 66: the generation of transgenic mice a
Page 67 and 68: CHAPTER 2 AIMS AND OBJECTIVES 67
Page 69 and 70: CHAPTER 3 MATERIALS AND METHODS 69
Page 71 and 72: Name of oligonucleotide EF1α Forwa
Page 73 and 74: cleavage sites GAAGGTATATTGCTGTTGAC
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R E1 A GAACGAATAGTGAAGCCACAGATGTA s
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100mm 25ug 225ul 250ul 500 1000ul T
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for the transgene were considered a
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solution in TBS-T containing 0.02%
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mounting agent. Confocal images wer
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3.26 Antibodies used for Western bl
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each plate was added cold 500ml of
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3.32 Hanging drop assay to determin
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3.37 Biodistribution of 18 F -FDG u
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2.5M NaCl 150mM 60ml Tween-20 (Sigm
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CHAPTER 4 RESULTS 95
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Figure 4.1.1 Generation of lentivir
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4.1.2 Generation of lentiviral vect
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transduction with the virus. Untran
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Figure 4.1.6: Application of bi-cis
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Figure 4.1.7: Application of bi-cis
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Figure 4.2.1: Infection of morulae
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Figure 4.2.2: Generation of transge
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Figure 4.2.3: EGFP-f expression in
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sequenced and the distribution amon
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4.3 Generation of 14-3-3γ knockdow
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numbering). Alignment was done usin
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presence of EGFP-f by performing po
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the mouse tested infected with vect
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Figure 4.3.7: Staging of 14-3-3γ k
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containing spermatozoa is on the Y-
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Figure 4.3.9: 14-3-3γ knockdown le
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Figure 4.3.11: 14-3-3γ knockdown i
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intercellular space (12, 110, 115,
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Figure 4.3.13: 14-3-3γ loss leads
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Figure 4.3.16: Localization of PKP3
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Figure 4.3.18: Reintroduction of sh
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A B 139
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from our laboratory demonstrated th
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Figure 4.3.21: Plakoglobin recruitm
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4.3.23 (A and B)). These proteins g
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the desmosomal proteins recruitment
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Figure 4.3.25: Generation of stable
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KIF5B, however its reduction does n
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compared to vector control cells. (
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4.4 Generation of 14-3-3ε knockdow
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into the interstitial spaces of the
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Figure 4.4.3: 14-3-3ε knockdown in
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Figure 4.4.4: Patchy hair loss in 1
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follicle formation marked by black
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Figure 4.4.7: Phenotypes in 14-3-3
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compared to WT lungs (indicated by
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compared to wild type (Figure 4.4.1
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and CD4 positive lyumphocyte number
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Figure 4.4.13: Bone marrow cells fr
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Figure 4.4.15: Generation of stable
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Figure 4.4.16: NIH3T3 cells lacking
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Figure 4.4.18: Generation of induci
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CHAPTER 5 DISCUSSION 181
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to generate transgenic animals expr
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transgenic research and the use of
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(reviewed in (152, 374)). Kinesins
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subtypes known to date. Type IV col
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Figure 5.1 Schematic representation
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loss of 14-3-3ε the T cell activat
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BIBLIOGRAPHY: 1. Abraham, R. T. 200
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29. Blomer, U., L. Naldini, T. Kafr
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58. Cheng, C. Y., and D. D. Mruk. 2
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90. Elia, A. E., P. Rellos, L. F. H
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118. Girard, F., U. Strausfeld, J.
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150. Herrmann, H., M. Haner, M. Bre
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and activation of a cyclin E-cdk2 c
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213. Liu, D., J. Bienkowska, C. Pet
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242. Moll, R., W. W. Franke, D. L.
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274. Pagano, M., R. Pepperkok, F. V
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305. Russell, L. D. 1978. The blood
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inhibitor of metalloproteases-1 in
Page 219 and 220:
364. Tsunekawa, N., M. Matsumoto, S
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397. Yashiro, M., N. Nishioka, and
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Research: Biology to Prevention to
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