Human artificial chromosomes (HACs) offer an alternative system which does not suffer from these disadvantages. In particular, these vectors have a functional centromere which enables the HAC to be stably maintained as a single-copy episome, without the risk of integration into the host genome. They also have a large cloning capacity, which allows entire genomic loci (including all their regulatory elements) to be used. A recent paper by Kim et al. (2011) has now used such a vector in a VHL-null human kidney cancer cell line (786-O) to demonstrate that it can correct for its VHL deficiency.

The authors first isolated the VHL gene from human genomic DNA using transformation-associated recombination cloning. The vectors were then constructed and transfected into CHO cells, and RT-PCR and fluorescence in situ hybridisation (FISH) were used to confirm the presence of the VHL-containing HAC within these cells.

Using microcell-mediated chromosome transfer, the VHL-HAC was transferred to VHL-null 786-O cells. FISH showed that this construct propagated autonomously without any integration into host chromosomes, and RT-PCR confirmed the expression of wild-type VHL within these cells. Moreover, when compared to controls, this HAC downregulated the protein levels of HIF2α, Cyclin D1 and Cdk1, which are known targets of VHL. Additionally, VHL-deficient 786-O cells have a more branched and invasive phenotype when supplemented with either hepatocyte growth factor or foetal bovine serum, but these phenotypes and their migration were substantially suppressed with the VHL-HAC.

This study made use of a HAC in which the centrosome can be inactivated, resulting in the loss of the HAC. This feature provides a useful phenotypic control for any HAC-associated alterations within the cells. Accordingly, centrosome inactivation and HAC loss led to the increased expression of HIF2α, Cyclin D1 and Cdk1, back to their original levels. Altogether, these results suggest that the VHL gene expressed from the HAC produces a functional protein which can correct for the VHL deficiency within these 786-O cells.

Much more work is necessary before HACs can be developed as a treatment for VHL, but a similar system could be tested in FLCN-null cells. Nevertheless, a number of BHD-associated gene therapy projects are currently in progress – to learn more, please do read our articles about Dr Justin Roth, Dr Richard Harbottle, Dr Laura Denby and Dr Suet-Ping Wong. Alternatively, join us at 4^th BHD Symposium in Cincinnati on 28^th-30^th March to hear more about the current state of BHD research in general. Registration and abstract submission are open, and further information about the Patient and Family sessions can also be found here.

• Kim JH, Kononenko A, Erliandri I, Kim TA, Nakano M, Iida Y, Barrett JC, Oshimura M, Masumoto H, Earnshaw WC, Larionov V, & Kouprina N (2011). Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proceedings of the National Academy of Sciences of the United States of America, 108 (50), 20048-53 PMID: 22123967

Dr Cash’s work focused on identifying the biological role of FLCN. In 2011, Cash et al. showed that FLCN was involved in the regulation of apoptosis through the TGF-β signalling pathway. Cells that lacked FLCN were resistant to cell death and this was thought to be due to the dysregulation of pro-apoptotic proteins. This paper has been reviewed in a previous blog post. Lim et al. (2012) have recently found FLCN, FNIP2 and AMPK to be involved in MNU-induced apoptosis, further highlighting the link between FLCN and cell death. Read more about this paper and how apoptosis could be implicated in the pathogenesis of BHD syndrome in last week’s blog post.

In addition to TGF-β signalling, Dr Cash worked on the link between FLCN and mTORC1 activity, demonstrating that downregulation of FLCN leads to mTOR inhibition (Hartman et al., 2009). Furthermore, renal tumours in mice carrying a FLCN mutation had low levels of phosphorylated S6, suggesting low levels of mTOR activity and adding to the debate on how FLCN affects mTOR signalling, which is described in more detail here.

Dr Cash submitted his PhD thesis, entitled ‘Molecular mechanisms of the Birt-Hogg-Dubé tumor suppressor’, in 2010 and has now moved to a postdoctoral position at the CNIO in Madrid where he is focusing on cellular aging and metabolism. Watch our video interview, filmed at the Third BHD Symposium, to learn more about Dr Cash and his BHD work. A transcript and audio-only file are also available for this interview.

To learn more about current BHD research and to meet researchers working on BHD syndrome, attend the Fourth BHD Symposium, to be held in Cincinnati on 28^th-30^th March 2012. The meeting will include sessions for patients and researchers. Registration and abstract submission are now open and more information can be found here. We hope to see you in Cincinnati!

• Cash TP, Gruber JJ, Hartman TR, Henske EP, & Simon MC (2011). Loss of the Birt-Hogg-Dubé tumor suppressor results in apoptotic resistance due to aberrant TGFβ-mediated transcription. Oncogene, 30 (22), 2534-46 PMID: 21258407
• Hartman TR, Nicolas E, Klein-Szanto A, Al-Saleem T, Cash TP, Simon MC, & Henske EP (2009). The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis. Oncogene, 28 (13), 1594-604 PMID: 19234517
• Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2011). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair PMID: 22209521

Please click here for more details.

We are looking forward to seeing many of you at the Fourth BHD Symposium, Cincinnati, 28th-30th March!

Co-immunoprecipitation experiments using mouse YT102 Mgmt^-/- cells, which lack the enzyme MGMT that repairs alkylated DNA, confirmed that FNIP2 interacts with both FLCN and AMPK. The authors then showed that siRNA knockdown of FLCN and AMPKα in this cell line suppressed their apoptotic response to MNU when compared to siRNA controls. They subsequently used compound C (a specific inhibitor of AMPK) to show that it also suppressed MNU-induced apoptosis when compared to untreated YT102 cells. AMPK is usually activated after MNU treatment, which is associated with an increased level of AMPKα phosphorylation. However, less of this activation was observed after compound C treatment.

This result was investigated further by treating YT102 cells with MNU, and assessing the levels of phosphorylated AMPKα over 72 hours. Here, Lim et al. (2011) saw that the levels of phosphorylated AMPKα gradually increased over time, but that this increase was abrogated by the siRNA-mediated knockdown of FLCN. In addition, KH101 Mgmt^-/- Fnip2^+/- cells, which were shown to be resistant to MNU-induced apoptosis by Komori et al. (2009), also showed no increase in phosphorylated AMPKα after MNU treatment.

To confirm that AMPK activation is involved in the induction of apoptosis, AICAR (a specific activator of AMPK) was added to YT102 and KH101 cells. This treatment increased the levels of AMPKα phosphorylation, as well as the amount of cell death detected by trypan blue staining in YT102 cells. However, no such increases were observed in the FNIP2-defective KH101 cells after treatment. To see if this cell death was attributable to apoptosis, mitochondrial membrane depolarisation (which is known to occur during apoptosis) was measured in these cells, and it could be seen that this depolarisation took place in the YT102 cells (but not the KH101 cells) after the addition of AICAR. Similar results were obtained with YT102 cells treated with AICAR and FLCN-siRNA, where less cell death, mitochondrial membrane depolarisation and AMPKα phosphorylation were observed when compared to siRNA controls.

Together, these results suggest that FLCN and FNIP2 play a role in AMPK activation and the induction of apoptosis by MNU. However, the exact mechanism by which this occurs is still unknown, and the elucidation of this process should help us to further understand the pathophysiology of BHD syndrome.

• Komori K, Takagi Y, Sanada M, Lim TH, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2009). A novel protein, MAPO1, that functions in apoptosis triggered by O6-methylguanine mispair in DNA. Oncogene, 28 (8), 1142-50 PMID: 19137017
• Lim TH, Fujikane R, Sano S, Sakagami R, Nakatsu Y, Tsuzuki T, Sekiguchi M, & Hidaka M (2011). Activation of AMP-activated protein kinase by MAPO1 and FLCN induces apoptosis triggered by alkylated base mismatch in DNA. DNA repair PMID: 22209521

Abstract submission is also open for the symposium and the deadline for abstracts is the 15^th February 2012. Submit an abstract here.

We look forward to seeing you in Cincinnati!

The symposium aims to be the best BHD meeting to date, with stimulating scientific discussion between experts from around the world. There will be a mixture of talks and posters, as well as time to exchange ideas and discuss collaborations with other members of the BHD community. A social dinner will take place on the evening of Thursday 29^th March. Highlights from last year’s meeting can be read here.

Invited speakers will include Dr W. Marston Linehan of the National Cancer Institute at the National Institutes of Health, Professor George Thomas of the University of Cincinnati,  Professor Kuniaki Seyama of Juntendo University, Professor Arnim Pause of McGill University and Professor Dr Maurice van Steensel of the University Hospital Maastricht. Talks will focus on all aspects of BHD syndrome, including the skin, lungs and kidneys, as well as both clinical and basic science. To find out more about these speakers, see our video interviews with Dr Linehan, Professor Pause and Professor Dr van Steensel.

Abstract submission is now open and the deadline for abstracts is 15^th February 2012. Abstracts may be submitted for both basic and clinical science presentations and researchers working on related conditions are also encouraged to submit an abstract. Abstracts can be submitted here and more details, including instructions for abstract submission, can be found here.

As with previous symposia, there will also be sessions for patients and family members. These will be led by a genetic counsellor who is an expert in BHD syndrome. Previous patient sessions have been very informative, allowing patients to learn more about BHD, meet other people affected by the syndrome, as well as talk to experts about their symptoms. Read a review of last year’s patient session here.

Registration and hotel information will be available soon so please check back. We will also update the webpage as more information on the programme is released. Don’t forget to get your abstracts in before 15^th February! We are looking forward to welcoming you to the Fourth BHD Symposium in March.

To find out more, download the latest version of the database here.

Registration will be open soon.

Dr Suet-Ping Wong, working in the lab of Dr Richard Harbottle at Imperial College London, was nominated for the Fairbairn young investigator award and gave a talk on FLCN gene therapy using S/MAR vectors (for more information see our lab profile). The Harbottle lab have successfully developed vectors which express FLCN, and have shown re-expression of FLCN in UOK257 cells using these vectors. Additionally, the corrected cells have been xenografted into mice, with promising results. It is hoped that future in vivo experiments will continue to develop this therapeutic product as a potential treatment for BHD syndrome.

The difficulties facing the development of gene therapy products for rare diseases were highlighted by Dr Janneke de Wal from Amsterdam Molecular Therapeutics. The small population of affected individuals means data sets are small and large geographical areas need to be covered in order to gain enough participants for rare disease clinical trials. In Dr de Wal’s work developing Glybera, a gene therapy product for the rare disease lipoprotein lipase deficiency, more academics were involved than clinical trial participants.

Dr Marco Ranzani from the San Raffaele Telethon Institute for Gene Therapy in Milan gave another interesting talk on using insertional mutagenesis to identify cancer genes. Although insertional mutagenesis is seen as an adverse effect of gene therapy, it can be exploited for cancer gene discovery. Dr Ranzani showed that vectors which target and disrupt the Pten and Cdkn2a genes induced hepatocellular carcinoma (HCC) in mice models. Using this technique, the Braf, Fign, Sos1 and Dlk1-Dio3 genes were found to be associated with HCC development. Insertional mutagenesis using gene therapy could be a valuable technique to identify cancer-associated genes that could be potential therapeutic targets.

Information on upcoming meetings that may be of interest to BHD researchers can be found on the Conferences and Events page. More information on the upcoming 4th BHD Symposium, to be held in Cincinnati, USA, 28^th-30^th March 2012, will also be available soon.

To find out more, download the latest version of the database here.