Host cell factors play a critical role in the establishment and maintenance of Kaposi’s sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.
Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman’s disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.
1. KSVH Cultivation and Infection of DMVEC
2. Total Rna Isolation and Production of Cdna from Mock and KSHV Infected DMVEC
3. Microarray Comparative Analysis of Mock and KSHV Infected DMVEC Cells
Our laboratory performs microarray analysis at the Vanderbilt University Medical Center Microarray Shared Resource Core Facility on a fee-for-service basis. We compare output files for transcriptional dysregulation of genes in KSHV infected DMVEC cells to mock infected control cells. Transcriptional dysregulated genes in KSHV infected cells that are relevant to angiogenesis, cell proliferation or that represent metastatic biomarkers are validated in vitro using KSHV infected DMVEC cells using real time PCR, Western blots, and dual IFA; results are compared to findings in archival KS tumor tissue.
4. Quantitative Rt-pcr Transcriptional Analysis Dysregulated Genes Found in Mock- and KSHV-infected DMVEC
5. Dual Labeled Immunofluorescence Analysis of KSHV Infected DMVEC Cells
6. Dual Labeled Immunohistochemistry on Ks Tissue and Ks Tissue Microarrays for KSHV Lana and the Endothelial Cell Marker Pecam-1/cd31
7. Gene Delivery by Adenovirus Transduction of DMVEC Cells
8. Representative Results
Figure 1. Primary DMVEC cells from Lonza Corporation were maintained in EBM-2 media and were infected with BCBL-1 virus at a moi of 0.01. After infection DMVEC cell monolayers were examined daily. Ten days post infection mock infected cells show a cobblestone-like morphology and KSHV infected cells exhibited a spindle cell type morphology characteristic of spindle shaped endothelial cells found infected with KSHV in KS tumor tissue. Photographs were taken with a NIKON TE 2000S microscope mounted with a CCD camera at a total magnification of 100X.
Figure 2. Dual labeled indirect immunofluorescence staining was performed on KSHV infected DMVEC cells at 10 days post infection. Infected cells were dual stained with a monoclonal antibody to KSHV LANA and a goat polyclonal antibody to a specific host cell protein found to be altered after KSHV infection. A mixture of secondary donkey anti-mouse and donkey anti-goat antibodies conjugated to rhodamine and FITC were added. Cells were examined with a Nikon TE2000S fluorescent microscope fitted with a CCD camera. Mounting media with DAPI was used to visualize nuclei. All photographs were taken at a total magnification of 200x. Infected cell expressing the virus LANA protein (rhodamine stained) localized to nucleus show reduced expression of the host cell specific protein (FITC stained) localized to cytoplasm.
Figure 3. IHC in KS tumor tissue for LANA and PECAM-1/ CD31. AIDS KS tumor tissue was stained by single and dual labeled IHC for KSHV LANA and PECAM-1/CD31. A) Shows KS tissue stained with a monoclonal antibody to LANA and counterstained with hematoxylin. LANA positive cells are identified by white arrows. B). KS tissue stained with a monoclonal antibody to PECAMI/CD31 and staining appears specific to endothelial around vessels depicted by white arrows. C) Shows KS tissue dual stained for LANA (vector red) and CD31 (brown) depicted by black arrows for LANA a white for cells staining positive for CD31. D) Shows a lower magnification of an area from the same slide in panel C. Brightfield images were photographed on a NIKON TE2000S microscope at total magnification of 600x A-C and 200x for Panel D.
Figure 4. Temporal expression of KSHV LANA by qRT-PCR 10 days post infection compared to mock infected control cells. All values for KSHV infected DMVEC cells were normalized to GAPDH. There was no evidence of LANA cDNA amplification in mock infected cells.
Figure 5. KS tumor tissue microarrays. The tissue microarray (TMA) represents 0.6 millimeter tissue cores arrayed on a single slide. Each tissue core represents a KS tumor specimen from a different patient. TMA were made available through the AIDS Cancer Specimen Resource (ASCR) from Leona Ayers M.D. at Ohio State University. Paraffin embedded KS tumor tissue along with matched normal and positive control tissues were placed on glass slides. KS tissue specimens were taken from mouth, skin, soft palate, tongue, and neck masses. All patients were HIV positive.
Figure 6. Tissue microarrays were stained for KSHV LANA by immunohistochemistry and were analyzed by brightfield microscopy. Color development for LANA was performed with DAB as a peroxidase substrate and LANA positive cells were visualized by an intense brown staining pattern. Different amounts of viral burden and spindle cell regions can be observed. A) represents focal viral infection in defined region of the tumor. B) represents disseminated infection with a high viral burden that is roughly correlated with the number of spindle cells present. Stained tissue cores were observed at 200x magnification.
Figure 7. DMVEC cells cultivated in EBM-2 complete media at a density of 2.5 X105 cells per well were transduced with 5 μL of Adenovirus at a titer of 1.0X1010. Cells were examined for EGFP fluorescence 18 hours post transduction. Both phase and fluorescent images were taken on a Nikon TE 2000S microscope at a total magnification of 200X. Transfection of primary DMVEC cells has proven to be problematic therefore we have identified viral vectors that will allow us to deliver fibulin-2 and galetin-3 very efficiently in primary DMVEC. We found that an adenovirus expressing EGFP can efficiently transduce primary DMVEC. We also found that the lentiviral vector we obtained from Lentigen Corporation was equally efficient for transducing primary DMVEC cells (data not shown).
The methodologies used in this report can be performed in a variety of laboratory settings. They require some special equipment or access to core facilities. Proper precautions must be taken when doing experiments involving infectious viral pathogens like KSHV which may require the use of a biosaftey level 3 (BSL-3) facilities. Tissue acquisitions must also be approved by the appropriate institutional review boards (IRBs).
These methodologies together can provide a basis for identifying genes that are dysregulated in expression in vitro and provide defined methods for in vivo validation of altered gene expression profiles as well strategies for gene delivery in endothelial cells. These findings taken together may help to elucidate biochemical pathways that are impacted by KSHV infection and subsequently by specific viral genes. An understanding of how oncogenic viruses like KSHV interrupt specific molecular pathways could provide insights in the development of novel therapeutics.
The authors have nothing to disclose.
We thank James E.K. Hildreth, Meharry Medical College, for his advice and review of this manuscript. We also thank Diana Marver for editing of the manuscript. This work was supported by the Meharry Vanderbilt Center for AIDS Research (NIH grant P30 AI054999-05); the Center for AIDS Health Disparities Research, NIH grant 5U54 RR019192-05; the Meharry Center for Clinical Research, NIH Grant P20RR011792; and by NIH grant P01 CA113239. D.J.A was partially funded by pilot grants from the Vanderbilt-Meharry Center for AIDS Research (CFAR) and the Meharry Center for Clinical Research (CRC).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
RPMI 1640 | Invitrogen | 05-0001DJ | ||
TPA | Sigma | P1585 | ||
PBS pH 7.4 | Invitrogen | 10010-023 | ||
Sodium butyrate | Sigma | 19364 (FLUKA) | ||
EBM-2 media | Lonza | CC-3156 | + bullet kits supplement | |
DMVEC cells (HMVEC) | Lonza | CC2543 | ||
Fetal calf serum | Hyclone | SH30066.03 | ||
Penicillin/streptomycin | Invitrogen | 15140-122 | ||
Chamber slides | Nalgene | 154461 | ||
KSHV LANA Mab | Vector Labs | VP-H913 | 1:50 dilution/IFA | |
Galectin-3 goat pAb | R&D Systems | AF1154 | ||
Donkey-anti-goat FITC | Jackson ImmunoRessearch | 705-095-1 | ||
Donkey-anti-goat Rho | Jackson ImmunoRessearch | 705-295-003 | 1:100 dilution/IFA | |
Mounting media | Vector Labs | H 1500 | Vectashield with DAPI | |
BCA Protein Assay Kit | Pierce | 23235 | ||
Nitrocellulose | Bio-Rad | 161-0112 | ||
Donkey-anti-goat-conj. | Santa Cruz | SC 2020 | ||
Western substrate | Pierce | 34075 | ||
Biotin-rabbit-anti goat | DAKO | 305-065-045 | ||
AP-Strepavidin conj. | DAKO | K 0492 (kit) | ||
RNase DNase Free Set | Qiagen | 79254 | ||
MJ Mini Cycler | Bio-Rad | PTC-1148C | ||
Bio-Photometer | Eppendorf | 952000006 | ||
RC 6 Plus Centrifuge | Sorvall | 46910 | ||
Coulter Optima L-90K Ultracentrifuge | Beckman | 392052 | ||
MYiQ iCycler Real Time PCR Detection System | Bio-Rad | 170-9770 | ||
TE 2000S Fluorescent microscope | NIKON | TE2000S | ||
2100 Bioanalyzer | Agilent | G2938C | ||
Eclipse E 200 | NIKON | E 200 | ||
Sorvall Legend RT Centrifuge | Thermo | 75004377 |