Transcriptional biomarkers and methods for detecting and isolating cancer cells in body fluids and tissue specimens
a transcriptional biomarker and cancer cell technology, applied in the field of detection and treatment of cancer, can solve the problems of increasing the cost of treatment, so as to extend the usefulness of the disclosed molecules and methods, and improve the effect of function
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example 1
Identification of Isoforms of Transcriptional Co-Regulators
[0123]This example demonstrates the results of an extensive in silico analysis of components of transcriptional co-regulators and use of PCR primers designed to identify novel iosforms with altered activity.
[0124]Material & Methods
[0125]Primary Tumors
[0126]Surgical specimens were obtained from human patients undergoing surgery for melanoma. Specimens were trypsinized and prepared for analysis using conventional techniques. RNA isolation was performed as described below.
[0127]Cell Culture
[0128]Human melanoma cell lines SK-MEL-28 and WM-266-4 were obtained from the American Type Culture Collection (ATCC; Manassas, Va.; SK-MEL-28 deposited by T. Takehashi and subject to release terms set by The Memorial Sloan-Kettering Cancer Center; WM-266-4 deposited by M. Herlyn). Cells were cultured according to recommendations of ATCC (DMEM, 10% FCS, penicillin+streptomycin) and used in experiments after two passages in the laboratory. Cel...
example 2
Effect of Interfering Peptides on Proliferation and Apoptosis of Melanoma Cells
[0140]Modeling of TFCs that contain isoforms of BAF57 and TRAP100 identified specific interactions that enabled us to synthesize small peptide libraries. Screening of these libraries using melanoma cell line SK-MEL-28 resulted in two peptides, denoted by us as BAF57 P12 and TRAP100 P05 that were found to stimulate apoptosis and inhibit growth of melanoma cells in vitro.
[0141]Amino acid sequences of SMARCE1 / BAF57 and TRAP100 isoforms. Unique, isoform specific sequences are underlined.
SMARCE1 / BAF57(SEQ ID NO: 10)MSKRPSYAPPPTPAPATQMPSTPGFVGYNPYSHLAYNNYRLGGNPGTNSRVTASSGITIPKPPKPPDKPLMPYMRYSRKVWDQVKASNPDLKLWEIGKIIGGMWRDLTDEEKQEYLNEYEAEKIEYNESMKAYHNSPAYLAYINAKSRAEAALEEESRQRQSRMEKGEPYMSIQPAEDPDDYDDGFSMKHTATARFQRNHRLISEILSESVVPDVRSVVTTARMQVLKRQVQSLMVHQRKLEAELLQIEERHQEKKRKFLESTDSFNNELKRLCGLKVEVDMEKIAAEIAQAEEQARKRQEEREKEAAEQAERSQSSIVPEEEQAANKGEEKKDDENIPMETEETHLEETTESQQNGEEGTSTPEDKESGQEGVDSMAEEGTSDSNTGSESNSATVEEPPTD...
example 3
Treatment of Human Melanoma Xenografts Using CSTC-Targeting Peptides
[0144]This example demonstrates the effect of therapeutic peptides on development of human melanomas in 4-week-old BALB / cOlaHsd-nu mice (Harlan, UK). Seven days after injection of melanoma cells, mice were randomly divided into 2 groups, 10 animals each. Control animals received intravenous (tail vein) injections of 50 microliters of phosphate buffer solution (PBS) every other day for 3 weeks. Test animals received intravenous (tail vein) injections of peptides BAF57 P12 and TRAP100 P05 (together) at a concentration of 0.5 mM each in 50 microliters of PBS every other day for 3 weeks. Last 2 injections were done using peptides labeled with fluorescein.
[0145]Therapeutic Peptides
BAF57 P12-PKKRKVRRRRRRRNDRLSDGDSKYSQTSHKLVQLLTRAP100 P05-PKKRKVRRRRRRRPQMQQNVFQYPGAGMVPQGEANFRed-NLS, blue-CPP and black-mimicking domains
[0146]One day following the last injection, animals were sacrificed and subcutaneous tumors were removed, ...
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