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MRC Prion Unit
From fundamental research to prevention and cure
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Molecular Cell biology

Prion Research

Cell lines have proven to be invaluable for in vitro studies of many complex processes and diseases including studying aspects of prion biology. The use of in vitro cell culture models to study prion propagation has been limited to a few cell-lines that are susceptible either to mouse-adapted or sheep scrapie prion strains, with none yet described able to stably propagate human prions. Long term studies at the MRC Prion Unit derived the cell line, PK1, a subline of neuroblastoma N2a cells, which efficiently propagate RML prions and which has allowed cell culture based bioassay for this strain of mouse prions1. However, this cell line is not permissive to many other prion strains.

Our studies are making use of this cell line to see if it was possible to use the strategy followed for making mice susceptible to prions from another species by inserting PrP’s from those species into Prnp0/0 mice. Since PK1 cells are polyploid and contain approximately 6 copies of PrP gene, we are using RNA interference to stably silence expression of endogenous Prnp in PK1 or parental N2a cells and then reconstitute them with the Open Reading Frames (ORFs) of prion protein genes of interest. We have developed a number of retrovirally delivered shRNAs located within the 3’UTR of mouse Prnp that are capable of silencing endogenous PrP and found that when two of them are used in combination PrPC expression can not be detected and results in loss of susceptibility to mouse prions.

Reconstitution using truncated and mutant mouse PrP molecules is being used to identify regions within PrP critical for high efficiency infection and propagation of mouse prions. Reconstitution of N2a null cells with ORF’s from other species is being attempted to develop cell lines permissive to other species of prions, notably human variant CJD prions.

We are also beginning to explore the use of induced pluripotent stem cell technology to develop iPS cells from patients to produce cellular models of inherited neurodegenerative diseases.

Dissecting the molecular mechanisms for the infinite proliferative potential of cancer cells

The central theme of this research is to identify the underlying molecular basis for the finite proliferative life span of normal somatic cells. Normal cells undergo a finite number of divisions and then cease dividing and undergo cellular senescence whereas cancer cells are able to proliferate indefinitely. This is triggered in response to a variety of intrinsic and extrinsic stimuli including alteration in telomere length and structure, DNA damage, physiological stresses and activation of oncogenes. It can compromise tissue repair and regeneration and contribute to tissue and organismal ageing due to depletion of stem/progenitor cell compartments. It can also lead to removal of defective and potentially cancerous cells from the proliferating pool thereby preventing neoplastic transformation and tumour development. The acquisition of an infinite proliferative potential is one of the six key events that are required for neoplastic transformation and one of the least understood since the underlying mechanism that controls cellular senescence and the signal transduction pathways involved are poorly understood. Such components should represent novel, important and direct targets for both cancer and anti-ageing therapies.

One of the main stumbling blocks for the slow progress in studying the finite proliferative life span has been the absence of suitable systems for study because of the asynchrony as well as the complexity of this process in heterogeneous cell populations. The discovery that certain viral oncogenes have the capacity to confer indefinite growth upon various cell types has allowed me to pursue a strategy analogous to the use of conditionally lethal mutants in the characterisation of many complex processes and develop reagents for conditional immortalization.

We developed the H-2Kbtsa58 strain of transgenic mice and showed that they can be used for the derivation of conditionally immortal cell lines from a wide variety of tissues that can undergo differentiation upon inactivation of LT antigen2. These include hippocampal cell lines that can be grafted into mice and permit recovery of spatial learning after ischaemia induced damage.

More recently we have developed reagents for conditional immortalisation of human cells and shown that freshly isolated human mammary fibroblasts, endothelial and luminal epithelial cells are not immortalised upon reconstitution of telomerase activity alone but additional activities were required which can be provided by SV40 LT antigen. Such immortalised cells remain dependent upon LT antigen to maintain their growth and it's inactivation results in a rapid irreversible cessation of cell growth3. Our finding that LT antigen interacts with Bub14, a spindle assembly checkpoint protein, has enabled us to derive immoral human cells that are diploid and exhibit long term karyotypic stability.

Work is now underway using these conditional cell systems to dissect the transcriptional networks that underlie cellular senescence by using expression profiling in conjunction with genetic complementation of the p53 and RB tumour suppressor pathways and gain of function RNA interference screens. A number of candidates have been identified which are now being analysed.

Breast Cancer

I also coordinate the Ludwig Institute for Cancer Research London Breast Cancer Initiative. In collaboration with Prof. Munro Neville, we have used Massively Parallel Signature Sequencing and four different array-based genome-wide methods to develop what is probably the most comprehensive catalogue of genes whose levels are altered in breast cancer cells and whose expression can be annotated with respect to whether they represent luminal or myoepithelial-type genes, free from the complexities due to the presence of normal and activated stromal cells present in solid tumour samples. The resulting differential tumour epithelial transcriptome consists of more than 8000 genes which are either up- or down-regulated in the malignant epithelial cells5. After ontological classification of these genes into different functional categories, we have focussed on transcription factors because they can significantly alter the biology of a cell by causing large scale changes in gene expression and may therefore be involved in many aspects of the malignant phenotype. 6 transcription factors that are consistently, highly differentially expressed in purified malignant versus normal epithelial cells have been identified; three of them are strongly down-regulated in tumours and tumour lines whereas three are up-regulated in the vast majority of tumours. Biological analysis of these transcription factors is now underway.

Recent Publications:

A systematic investigation of production of synthetic prions from recombinant prion protein.
Schmidt C, Fizet J, Properzi F, Batchelor M, Sandberg M, Edgeworth J, Afran L, Ho S, Badhan A, Linehan J, Brandner S, Hosszu L, Tattum H,  Jat  P, Clarke T, Klohn P, Wadsworth J, Jackson G, Collinge J. Open Biology 2015; in press

Novel highly specific anti-periostin antibodies uncover the functional importance of the fascilin 1-1 domain and highlight preferential expression of periostin in aggressive breast cancer. FieldS, Uyttenhove C, Stroobant  V, Cheou P, Donckers D, Coutelier J-P, Simpson PT, Cummings MC, Saunus JM, ReidLE, Kutasovic JR, McNicol AM, Kim BR, Kim JH, Lakhani SR, Neville AM,  Van Snick J, Jat PS.International Journal of Cancer 2015; in press.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance. Saunus JM, Quinn MC, Patch AM, Pearson JV, Bailey PJ, Nones K, McCart Reed AE, Miller D, Wilson PJ, Al-Ejeh F, Mariasegaram M, Lau Q, Withers T, Jeffree RL, Reid LE, Da Silva L, Matsika A, Niland CM, Cummings MC, Bruxner TJ, Christ AN, Harliwong I, Idrisoglu S, Manning S, Nourse C, Nourbakhsh E, Wani S, Anderson MJ, Fink JL, Holmes O, Kazakoff S, Leonard C, Newell F, Taylor D, Waddell N, Wood S, Xu Q, Kassahn KS, Narayanan V, Taib NA, Teo SH, Chow YP, kConFab, Jat PS, Brandner S, Flanagan AM, Khanna KK, Chenevix-Trench G, Grimmond SM, Simpson PT, Waddell N, Lakhani SR. J Pathol. 2015 Nov;237(3):363-78. doi: 10.1002/path.4583. Epub 2015 Aug 19. PMID: 2617239

Effects of CT-Xp Gene Knock down in Melanoma Cell Lines

Caballero OL, Cohen T, Gurung S, Chua R, Lee P, Chen YT, Jat P, Simpson AJ
Oncotarget. 2013 Apr;4(4):531-41




The expression of podocyte-specific proteins in parietal epithelial cells is regulated by protein degradation

Guhr SS, Sachs M, Wegner A, Becker JU, Meyer TN, Kietzmann L, Schlossarek S, Carrier L, Braig M, Jat PS, Stahl RA, Meyer-Schwesinger C.
Kidney Int. 2013 Apr 24. doi: 10.1038/ki.2013.115




Receptor-mediated endocytosis and endosomal acidification is impaired in proximal tubule epithelial cells of Dent disease patients

Gorvin CM, Wilmer MJ, Piret SE, Harding B, van den Heuvel LP, Wrong O, Jat PS, Lippiat JD, Levtchenko EN, Thakker RV
Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):7014-9. doi: 10.1073



An RNA interference screen for identifying downstream effectors of the p53 and pRB tumour suppressor pathways involved in senescence.

Guhr SS, Sachs M, Wegner A, Becker JU, Meyer TN, Kietzmann L, Schlossarek S, Carrier L, Braig M, Jat PS, Stahl RA, Meyer-Schwesinger C
Kidney Int. 2013 Apr 24. doi: 10.1038/ki.2013.115 


Activation of nuclear factor-kappa B signalling promotes cellular
senescence

E Rovillain, L Mansfield, C Caetano, M Alvarez-Fernandez, OL Caballero, RH Medema,
H Hummerich and PS Jat

Oncogene - 2011 Jan 611 


Dissecting the transcriptional networks underlying breast cancer: NR4A1 reduces the migration of normal and breast cancer cell lines

Alexopoulou AN, Leao M, Caballero OL, Da Silva L, Reid L, Lakhani SR, Simpson AJ, Marshall JF, Neville AM, Jat PS. 
Breast Cancer Res. 2010 Jul 19;12(4):R51


Conditionally immortalized human podocyte cell lines established from urine 

Sakairi T, Abe Y, Kajiyama H, Bartlett LD, Howard LV, Jat PS, Kopp JB  
Am J Physiol Renal Physiol. 2010 Mar;298(3):F557-67.


Decreased poly(ADP-ribosyl)ation of CTCF, a transcription factor, is associated with breast cancer phenotype and cell proliferation

Docquier F, Kita GX, Farrar D, Jat P, O'Hare M, Chernukhin I, Gretton S, Mandal A, Alldridge L, Klenova E. 
Clin Cancer Res. 2009 Sep 15;15(18):5762-71


Identification of differentially expressed sense and antisense transcript pairs in breast epithelial tissues

Grigoriadis A, Oliver GR, Tanney A, Kendrick H, Smalley MJ, Jat P, Neville AM. 
BMC Genomics. 2009 Jul 17;10:324

Simian virus 40 large T antigen disrupts genome integrity and activates a DNA damage response via Bub1 binding

Hein J, Boichuk S, Wu J, Cheng Y, Freire R, Jat PS, Roberts TM, Gjoerup OV.
J Virol. 2009 Jan;83(1):117-27


A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions.
Klohn P, Stoltze L, Flechsig E, Enari M, Weissmann C.
Proc.Natl.Acad.Sci U.S.A 2003;100(20):11666-71.

Direct derivation of conditional immortal cell lines from an H-2KbtsA58 transgenic mouse.
Jat P.S., Noble, M.D., Ataliotis , P., Tanaka, Y., Yannoutsos,N., Larsen, L. and Kioussis, D. 
Proc. Natl. Acad. Sci USA 1991. 88: 5096-5100.

Conditional immortalization of freshly isolated human mammary fibroblasts and endothelial cells.
O'Hare M. J, Bond J., Clarke C., Takeuchi Y., Atherton A.J., Berry C., Moody J., Silver A.R.J., Davies D.C., Alsop A.E., Neville A.M. and Jat P.S.  
Proc. Natl. Acad. Sci. USA 2001. USA.98: 646-651.

SV40 large T antigen targets the spindle assembly checkpoint protein Bub1.
Cotsiki M., Lock R. L., Cheng Y., Williams G. L., Zhao J., Perera D., Freire R., Entwistle A., Golemis E., Roberts T. M., Jat P. S. and Gjoerup O. V. 
Proc. Natl. Acad. Sci. USA 2004 101: 947-952.

Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.
Grogoriadis A., Mackay A., Reis-Filho J. S., Steele D., Iseli C., Stevenson B., Jongeneel C.V., Valgeirsson H., Fenwick K., Iravani M., Leao M., Simpson A. J. G., Strausberg R. L., Jat P. S., Ashworth A., Neville A. M., and O’Hare M.J. 
Breast Cancer Research 2006 8:R56. 

MRC Prion News