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PROTEOMICS TECHNOLOGY

The Fisher Biomarker Laboratory has applied a variety of methods to isolate, identify and characterize new biomarkers.  We have used several types of proteomics technologies for the discovery and clinical characterization of new biomarkers.  These biomarker discovery tools include applying Surface Enhanced Laser Desorption Ionization (SELDI) time of flight (TOF) or SELDI-TOF Mass Spectroscopy technology from Ciphergen (Now owned by BioRad Inc.), 1- Dimensional and 2-Dimensional Electrophoresis, and capillary electrophoresis from BioRad Laboratories.  Our work in the field of SELDI-TOF Mass Spectroscopy has been under collaborations conducted with the Eastern Virginia Medical School (EVMS) Center for Biomedical Proteomics with John Semmes and members of his staff and also with Dr. Daniel Chan of the department of clinical pathology at JHU School of Medicine.   The SELDI-TOF technology is illustrated below and includes the instrument, specially engineered “Chips” to analyze various biological matrices and a cartoon also illustrated below demonstrates the general principal of SELDI-TOF.


SELDI-TOF TECHNOLOGY

SELDI-TOF Mass Spectrometer

The Solid Phase Chip Surface Arrays

Chemical Surfaces
       My+
NR+3
SO-4
Hydrophobic
Ionic
IMAC-3
Mixed

Biochemical Surfaces
Antibody
DNA
Enzyme
Receptor
Drug
         

PRINCIPAL OF SELDI-TOF CHIP ASSAYS

PRINCIPAL OF MASS SPECTROSCOPY

Also, the Fisher Biomarker Laboratory has utilized two other proteomic technologies to discover and characterize new molecular biomarkers where the abundance and complexity of the molecules vary in concentration and structure.  One such tool is the use of Capillary Electrophoresis and the other uses 2-Dimensional (2-D) electrophoresis to isolate to candidate biomarkers of lower concentration and molecular complexity as seen below.  Additionally, the Fisher Biomarker Laboratory has the capability to purify new proteomic biomarkers using the BioRad automated Duo-Flow chromatography system also illustrated below.

CAPILLARY ELECTROPHORESIS TO CHARACTERIZE PROTEIN BIOMARKERS

 

ISOLATION OF NEW TUMOR BIOMARKERS USING 2-D

PURIFICATION OF PROTEINS BASED UPON MOLECULAR SIZE AND CHARGE

To illustrate applications of some of these technologies we have a few examples.  One of these projects involved the discovery of new biomarkers for PCa metastasis using the Dunning rat prostate tumor model and the SELDI-TOF instrument and the new BioRad Experion capillary electrophoresis system. We have discovered several proteins with BioRad’s SELDI-TOF, Chip-based Mass Spectroscopy.  Applying this technology our laboratory differentiated the three Dunning rat cell sublines based upon protein concentration normalized profiles between 5,000 and 20, 000 Daltons. The tumor cell preparations from the three Dunning PCa cells lines showed clear differences when the cell line extracts from the metastatic sublines (AT-1 and MLL) were compared to the benign subline (G) for proteins with molecular weights of 9 kD (decrease), 12 kD (significant decrease), 14 kD (decrease), and 17 kD (significant gain). After pre-processing extracts with ammonium sulfate and molecular ultrafiltration, the molecular profile changes from one subline to the next became more apparent. Our results were reproducible using multiple runs including from Dunning cells cultured in a separate lab, and using different lots of SELDI ProteinChips.

Next, we are focused identification of one of the three prior biomarkers discovered in the above experiments.  The cell line preparations from the three Dunning sublines (G, AT-1 and MLL) clearly demonstrated the original overexpressed ~17 kDa protein peak identified by Gretzer et al (Prostate 2004; 60:(4):325-31).  We therefore isolated the ~17 kDa proteins overexpressed in the metastatic cell lines, and subsequently identified two proteins that migrated in this ~17 kDa molecular fraction by Liquid Chromatography and Mass Spectroscopy (LC-MS/MS); the predominant protein identified was Histone 2B and a second was Adiponectin (rat Adipocyte lipid-binding protein). The H2B protein was identified in collaboration with EVMS and H2B is known to control gene transcription from DNA through chromatin remodeling.

WHERE ARE THE HISTONE PROTEINS LOCATED AND WHAT DO THEY DO?

H2B is one of four members of the Histone Tail Code family shown above that are involved in Nuclear Chromatin remodeling at the level of gene transcription.  When appropriate a complex of DNA binding transcription factors bind to DNA and they will initiate transcription through opening of the DNA chromatin structure so that the DNA can initiate transcription of the genes being activated.  Clearly, this molecular step in the pathology and pathogenesis of cancer is important and other scientists have verified it relevance to recurrence and progression of this devastating disease.

SELDI-TOF IMMUNOASSAY: INCREASE OF H2B IN METASTATIC DUNNING TUMORS

The Figure to the left illustrates the Detection of Histone H2B levels using SELDI immunoassay. Total cell extracts of G, AT-1 and MLL cell lines were tested for Histone H2B levels using the SELDI-based immunoassay with H2B antibody coated PS20 ProteinChip®arrays. (A)Gel view of the spectra obtained from anti-H2B coated PS20 ProteinChip®arrays on SELDI-immunoassay. (B) Gel view of the spectra obtained from anti-acetylated H2B coated ProteinChip®arrays on SELDI-immunoassay. Arrow points to the ~17.5K m/z protein peak differentially captured by the antibodies. About 12 fold increase in H2B and 4 fold increase in acetylated H2B in MLL samples is observed as compared to G cell lines (C). MLL extracts were incubated on anti-PSA coated spots as controls (Ctrl.).

 

PBOV1 (UROC28): Molecular Characterization a Novel Molecular Marker for Prostate Cancer
A novel membrane protein (p17) coded for by the PBOV-1 (UROC28) gene is overexpressed in prostate, breast, and bladder cancers (Cancer Research 60: 7014-7020, 2000).  PBOV-1 is encoded by a single copy of gene at chromosome 6q23-24.  The increased expression of PBOV-1 mRNA correlates with increasing Gleason Score and advancing pathological stage of disease.  We investigated the expression of PBOV-1 protein in PCa cell lines and sera and tissue from PCa patients both with low and high metastatic potential.  The above immunoassay approach has also been applied to other cancer biomarkers being studied by the Fisher Laboratory.  Once such protein is the prostate breast overexpressed gene-1 (PBOV-1) protein that is overexpressed in PCa and Breast cancer and the example below demonstrated the detection of PBOV-1 in the serum of patients with BPH and PCa. Three different antibodies to the PBOV-1 protein were tested below (Anti- C, -B and –A) and the Anti-B PBOV-1 antibody had the best sensitivity and specificity.  Differential expression of PBOV-1 between low and high metastatic potential was detected in PCa cell lines, PCa human sera and tissues.  Further molecular characterization of PBOV-1 may lead to the development of a serum immunoassay and tissue with potential clinical diagnostic and prognostic value for PCa.  Work is continuing on this biomarker to determine if it may be useful to supplement the Prostate Specific Antigen test for the detection and monitoring of PCa.  The Figure A below illustrates the ~17 kDa PBOV1 protein expression in the Dunning PCa cell lines MLL (100% metastatic), AT6 (20% metastatic) and G (0% metastatic).  Figure B illustrates the PBOV1 protein in human sera from 10 cases each of normals, BPH, and PCa cases comparing PBOV1-A/C specific rabbit antisera.

Anti-PBOV1-B Antibody Type Detects the ~17 kDa in PCa Cell Lines with various Metastatic potentials


Anti-PBOV1-A vs. Anti-PBOV1-C and A Antibody Types (~17 kDa in Human Serum)


A NEW Anti-B PBOV1 antibody – We have immunostained Benign prostate and Gleason scores (GS) 3+3 and 4+4 PCa tissues with affinity-purified rabbit PBOV1 Anti-B (AA 1-19). We incubated a U1:500 dilution of the Anti-B PBOV1 antibody overnight at 4C and then completed the Avidin-Biotin Complex HRP method. Note the IHC differences in areas & intensity of sSpecific PBOV1 immunostaining of the membrane (B & C) Uof the cancer epithelial cells. This new Anti-PBOV1-B epitope could be of prognostic value for PCa in biopsies.

 BLADDER CANCER: PROTEOMICS & IMAGE ANALYSIS:
Dr. Sumit Isharwal, Post-Doctoral Fellow from the All India Institute of Medical Sciences (A.I.I.M.S.), New Delhi, India.  In one areas of his research, he has used 2D electrophoresis described above to identify new candidate biomarkers for BlCa, transitional cell as well as squamous cell carcinoma, by characterizing two cell lines respectively.  The TCC cell line is Tccsup and the SCC cell line is SCaBER cell line and comparisons were made to a normal bladder endothelial cell line (HBdMEC-p).  


Current 2D Proteomics Experience in Bladder Cancer (Dr. Veltri):
In the figure below we demonstrate preliminary results for 2D PAGE comparing normal and a TCC versus a SCC cell line solublized extracts.

Dr. Isharwal prepared the HBdMEC-p (normal endothelial bladder cell line) in endothelial cell growth media; the Scaber (a squamous cell carcinoma, SCC cell line) and transitional cell carcinoma superficial (TCCSUP) were grown in MEM media with 2mM L-glutamine, Earle’s BSS, 0.1mM non-essential amino acid, 1mM sodium pyruvate and 10% FBS were grown and harvested mechanically at 80% confluency.  The Fisher Laboratory prepared whole cell line lysates using 7M urea, 2M thiourea, 4% CHAPS, 2% Biolyte 3/10 as the lysis buffer.  Next, extracted samples were post-processed using BioRad 2D clean up kit, and total protein was quantified using BioRad RC-DC assay. A total of 50 μg protein was dissolved in 350 µL rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 2% Biolyte 3/10, 1.5% 2-hydroxyethyl disulfide, trace of bromophenol blue) and then incubated passively overnight with pH 5-8 IPG strip. Isoelectric focusing with 75,000 v-hr using BioRad protean IEF cell system and 2nd dimensional separation was analyzed using precast 17cm gels. By visually comparing Normal, Scaber and TCCSUP cell line 2D gel, there were several gel spots present only in Scaber 2D gel and we are pursuing these as possible targets .

Also, in the area of BlCa, the Fisher Biomarker Lab, under is studying the etiopathogenesis of Schistosoma haematobium infection and development of BlCa in an endemic area of Accra, Southern Ghana. This project is an active collaboration between Dr. Clive Shiff in the Bloomberg School of Public Health and Dr. Robert Veltri in the Brady Urological Research Institute in the Johns Hopkins School of Medicine. We are also collaborating with Dr. Bosompem who is our principal investigator at the Noguchi Memorial Institute for Medical Research in Accra, Ghana.  Dr. Naples and her collaborators in Ghana examined and obtained urine specimens from 576 volunteers in three endemic villages in Ghana (Chento, Ayiki Doblo and Ntoaso), and we have found high infection rate of adults for this parasite living in this highly endemic area. Furthermore, in a significant percentage of 378/576 volunteer cases tested using a portable ultrasound instrument we observed moderate to severe bladder damage when applying the WHO criteria. These techniques were perfected in collaboration with the ultrasound technologists in the department of Radiology in the School of Medicine and guideline published by WHO. Also using a portable ultrasound machine, Our findings show that between 69-94% of adults examined in each village have abnormal bladder lesions (bladder masses, polyps, wall calcification/abnormal shape and hydronephrosis) including those who are negative for current infection by our testing criteria but give a verbal past history of urinary schistosome infection.  In addition, we assessed BLCA-4 protein biomarker (a nuclear matrix protein found in urine of bladder cancer patients), by direct ELISA in 194 cases of those who had ultrasound scans and about ~30-40% of cases with severely abnormal ultrasound cases had positive results with BLCA-4.

More recently, Dr. Naples has demonstrated the contribution of nuclear morphometry of the transitional and squamous epithelial cells found in the urine of volunteers from Ghana being evaluated for severe bladder abnormalities as a result of schistosomiasis infections.  Note, the squamous type cells had the best diagnostic outcome.  In fact, a logistic regression model constructed using a QNG solution of squamous cells in the urine produced a patient-specific prediction for normal vs. abnormal bladder pathology and yielded an AUC-ROC of 86.90% and at a cutoff of 0.40 for the predictive probabilities, the diagnostic sensitivity, specificity and accuracy were 85.00%, 76.19% and 80.5 to detect abnormal bladder pathology caused by Schistosoma pathogens.

 

Dr. Jean Naples, M.D., Ph.D. is a post-doctoral Fellow Studying Bladder Cancer in Ghana, Africa.  Dr. Naples prior experience as a Peace Corp volunteer in Mauritania in 1977 followed by her medical technology experience, academic pursuits at U.C. Berkeley, medical and surgical training and a PhD in the School of Public Health at Johns Hopkins has provided a solid basis for her success. Also, her prior research experience in the field of Schistosomiasis involved the mechanism of transmission of the disease mouse model.    Jeans has made several trips to Accra, Ghana to study the epidemiology and the pathogenesis of Schistosoma infections and its potential to cause bladder cancer.    Jean has developed a very strong working relationship with the Noguchi Memorial Medical Research Institute (NMIMR) in Noguchi, Ghana as well as the Korle bu Hospital in Accra. 


Below are examples of field ultrasounds collected by Jean in one of the villages along the Densu River and you will note the abnormal observations.  Also, below is urine cytology samples collected and stained by PAP and Feulgen cytochemical stains. On the left are abnormal field ultrasounds performed by Dr. jean Naples and the Red Arrows indicate abnormalities.  On the right urine cytology slides illustrate Schistosomes and they are marked by the yellow arrows.

Abnormal Field Ultrasounds Obtained in Ghana 

Schistosomes in urine specimens from Ghana





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