CANCER GENOMICS & PROTEOMICS
Volume 2, Number
6, November-December 2005
CONTENTS
PAGE
Genomic Instability, DNA Alterations and Tumor Eosinophilic Expression in Head and Neck Squamous Cell Carcinoma.S.J. ALRAWI, D. STOLER, D. TAN, M. DAYTON, T. LOREE, J.F. GIBBS, N. RIGUAL, S. SAIT, T. KHOURY, W. HICKS JR, G. ANDERSON (Buffalo, NY, USA)
307
Quantitative Real-time RT-PCR: Application to Carcinogenesis. F.E. AHMED (Greenville, NC, USA)
317
Tissue Profiling by MALDI Mass Spectrometry Distinguishes Clinical Grades of Soft Tissue Sarcomas. R.L. CALDWELL, G.E. HOLT, R.M. CAPRIOLI (Nashville, TN, USA)
333
Proteomic Analysis of a Human Prostate Cancer Cell Line after Incubation with a Novel Somatostatin14 Derivative. Z. LIU, M. M?RQUEZ, S. NILSSON, A.R. HOLMBERG, A. ALAIYA (Stockholm, Sweden; Jinan, China)
347
Tumour-associated Proteins in Oral Squamous Cell Carcinomas by Proteomics. S.O. IBRAHIM, T.MIRON, M. KROHN, A.N. AMARATUNGA, S. WARNAKULASAURIYA, E. N. VASSTRAND (Bergen; Oslo, Norway; Rehovot, Israel; Peradeniya, Sri Lanka; London, UK)
353
*Reviews
(pages 317)
Volume 2, 2005, Index
365
CANCER GENOMICS & PROTEOMICS 2: 347-352 (2005)
Proteomic Analysis of a Human Prostate Cancer Cell Line after Incubation with a Novel Somatostatin14 Derivative
ZHAOXU LIU1,2, MARCELA MARQUEZ1, STEN NILSSON1, ANDERS R. HOLMBERG1, AYODELE ALAIYA1
1Karolinska Institute, Urologic Oncology Group, CCK R8/3, 17176 Stockholm, Sweden; 2School of Nursing, Shandong University, Jinan, 250012,China
Abstract: Background: Derivatives of somatostatin (sms) are sometimes used in the treatment of hormone-refractory prostatic cancer, in spite of modest results in controlled clinical studies. The optimal use in this context remains to be determined. The human prostatic cancer cell line LNCaP has been used in several previous proteomic analysis studies, which confirmed that sms indeed can affect the protein expression in this cell line. Proteomic analysis is an important tool to increase the understanding of how sms affects the protein expression of the tumor cell. Materials and Methods: In this in vitro study, a new sms14 derivative, smsdx, a conjugate between sms14 and dextran, was incubated with an LNCaP cell culture. Sms14 was used as the positive control. Proteomic analysis, using rapid mini two-dimensional electrophoresis, was performed to determine the effects on protein expression. Results: Marked quantitative differences were observed in the protein expression profiles in smsdx-treated LNCaP cells compared to negative control cells (untreated cells). Sets of 63 (yet unidentified) protein spots were differentially expressed. The difference was statistically significant (Mann-Whitney analysis). The 63 dataset was used to accurately discriminate control cells from smsdx-treated cells using hierarchical cluster analysis. Both similarities and differences in protein expression were observed between smsdx- and sms14-treated cells. Conclusion: Smsdx is a new sms14 derivative with long in vivo half-life and pan receptor affinity. Sms14-like effects on the protein expression of LNCaP cells seem to be preserved in the construct. The results convey new information about this potentially useful compound. Further studies are now in progress to identify the affected proteins
Genomic Instability, DNA Alterations and Tumor Eosinophilic Expression in Head and Neck Squamous Cell Carcinoma
SADIR J. ALRAWI1, DANIEL STOLER2, DONGFENG TAN3, MERRIL DAYTON4, THOM LOREE1, JOHN F. GIBBS1, NESTOR RIGUAL1, SHEILA SAIT3, THAER KHOURY3, WESLEY HICKS JR1, GARTH ANDERSON2
Departments of 1
Head and Neck/Surgical Oncology, 2Cancer Genetics and 3Pathology, Roswell Park Cancer Institute, Buffalo, New York; 4Department of Surgery, University of Buffalo, SUNY, New York, U.S.A.
Abstract: The progression of normal cells to invasive tumor cells has been attributed to the acquisition of numerous mutations in the genome; genomic instability (GI) facilitates the accumulation of these mutations. To study the GI in head and neck squamous cell carcinoma (HNSCC), the genomic instability index (GII) was examined; chromosomal gains and losses were evaluated by array comparative genomic hybridization (aCGH), which were confirmed by fluorescence in situ hybridization (FISH). Histopathological eosinophilic infiltrate (Eos Infil), as an adjunct measure of tumor invasiveness, was also considered and compared to GI. Materials and Methods: Inter-simple sequence repeat PCR (ISSR-PCR) was utilized to determine the GII (a quantitative estimate of the relative overall genomic damage). GII was measured in 26 pairs of tumor and normal HNSCC samples. Array CGH was conducted using bacterial artificial chromosome (BAC) clones to evaluate amplifications and deletions (n=20 tumor), and confirmation of the specific changes was made by FISH analysis. Histopathological evaluation of Eos Infil for all samples was performed to calculate the eosinophilic index (EI). This was accomplished by observing Eos Infil in neoplastic tissue and at the tumor/normal tissue interface. Results: GI was evident in 25 of the 26 tumors. The GIIs ranged from 0 to 5.3% with a mean of 2.8% (similar to the results reported for colorectal and thyroid carcinomas). GIIs were higher in tumors of the oral cavity (OC) than in tumors from other subsites of HNSCC (p=0.05). Chromosomal alterations were identified by array CGH on chromosomes 8, 11 and 17, but consistent amplifications were observed on (8q21.3-23.5) in 80% of the tumors. These changes were confirmed using FISH analysis. There was an association between increased GI and rising EI, but this did not achieve statistical significance (p>0.05). Conclusion: HNSCC exhibits a similar degree of GI to that previously reported in colorectal and thyroid malignancies. The higher GI identified in OC tumors could be explained by a greater degree of the damage from environmental carcinogens (smoke, diet and alcohol) to the first station of the areodigestive tract. Consistent amplification at the specific loci of 8q might be attributed to mutations in various genes, such as SHAX3 (Snf7 homolog, associated with Alix 3, involved in apoptosis and endocytosis) and E2F5 (transcription factor 5 that interacts with tumor suppressor proteins). EI was not associated with age, sex, site or stage of tumor in established cases. Further work up will be necessary to explain these results.
Quantitative Real-time RT-PCR: Application to Carcinogenesis
FARID E. AHMED
Department of Radiation Oncology, Leo W. Jenkins Cancer Center, The Brody School of Medicine at East Carolina University, Greenville, NC, U.S.A.
Abstract: Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) has simplified and enhanced the quantification of gene expression. However, since no agreed standardizations are available, care must be exercised when designing experiments, including the choice of appropriate amplification primers, detection chemistry and the normalization procedure, in order to obtain meaningful results. Coupling quantitative polymerase chain reaction (qPCR) to cell purification from tumor tissue has made it possible to decrease the variability in expression from in vivo heterogeneous cell populations. Sensitive and specific qRT-PCR has advanced the diagnosis, prognosis and prediction response of colorectal cancer to therapy.
Tumour-associated Proteins in Oral Squamous Cell Carcinomas by Proteomics
SALAH OSMAN IBRAHIM1, TALIA MIRON2, MARIT KROHN3, ASOKA N. AMARATUNGA4, SAMAN WARNAKULASAURIYA5, ENDRE NORMANN VASSTRAND6
1Department of Biomedicine, Section of Biochemistry and Molecular Biology,Jonas Lies Vei 91, 5009 Bergen, University of Bergen, Norway; 2Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel; 3Department of Cell Biology, Institute for Cancer Research, University of Oslo, Norway; 4Division of Oral Surgery, Dental School, University of Peradeniya, Sri Lanka; 5Guy's, King's and St. Thomas' Dental Institute, King’s College, London, U.K.; 6Faculty of Oral Sciences –Periodontology, Årstadveien 17, 5009 Bergen, University of Bergen, Norway
Abstract: Antibody microarrays, two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (2-DE/MALDI-TOF-MS) were used to examine protein changes in 56 oral cancers (OCs)/normal controls (NCs) from Sudanese (41 OCs vs. 31 NCs) and Sri Lankan (15 OCs vs. 15 NCs) patients. Pools of extracted proteins were prepared and used for microarrays/2-DE/MALDI-TOF-MS. From 2-DE, protein spots (differentially- expressed) were cut and identified with peptide mass fingerprinting based on MALDI-TOF-MS, and the proteins were identified by submitting peptide mass profiles to the NCBInr database. By microarrays, 6 and 8 proteins demonstrated significant differences in their abundance values as differentially-expressed in OCs examined from Sudan and Sri Lanka, respectively. For some of the proteins found, like p56dok2 and NEK2, this is the first report in OCs. By MALDI-TOF-MS/2-DE, patterns of OCs/NCs were acquired and tumour-associated proteins, like psoriasin, calgranulin-B and glutathione transferase, were found to be altered in OCs compared to NCs. The proteins found in this work (by two different methods) represent a global protein change specific to OCs from two populations. This might indicates involvement of multiple pathways in the process of tumorgenesis; thus, multiple proteins should be simultaneously targeted in OCs. The finding of few common proteins might suggest involvement of different pathways, which may parallel differences in ethnicity and/or lifestyle.
Tissue Profiling by MALDI Mass Spectrometry Distinguishes Clinical Grades of Soft Tissue Sarcomas
ROBERT L. CALDWELL1, GINGER E. HOLT2, RICHARD M. CAPRIOLI1
1Mass Spectrometry Research Center and 2Department of Orthopaedics and Rehabilitation, Vanderbilt University School of Medicine, Nashville, TN, U.S.A.
Abstract: Soft tissue sarcomas are rare, heterogeneous, mesenchymal tumors that have been poorly classified. The heterogeneity of these aggressive tumors has made consistent tumor grading difficult and accurately predicting the behavior of the tumor based on the histological subtype and grade has been challenging. Molecular profiling of proteins in tissues may present a new avenue for distinguishing clinical grades and overall tumor aggressiveness. Direct tissue analysis using matrix-assisted laser desorption ionization mass spectrometry is a new technology that permits rapid detection of hundreds of proteins from intact tissues. We have employed this technology to profile human soft tissue sarcomas to discover protein biomarkers that distinguish tumor grade. Profiling was accomplished on histologically-stained tissue sections, allowing highly reproducible spectra for each sarcoma grade to be obtained. Forty-two tissue specimens were analyzed in this manner. Several proteins specific for high-grade sarcomas were identified and confirmed with immunohistochemistry. Proteins present in control tissue, that are suppressed by soft tissue sarcomas, were also identified.
