Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins.
The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.
Lange
Andreas
A
German Cancer Research Center, Skin Cancer Unit, Heidelberg, Germany.
Kistler
Claudia
C
Jutzi
Tanja B
TB
Bazhin
Alexandr V
AV
Klemke
Claus Detlev
CD
Schadendorf
Dirk
D
Eichmüller
Stefan B
SB
D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology*; D004072:Digitonin / Q000494:pharmacology*; D018870:Endoplasmic Reticulum, Rough / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D052138:Genes, Neoplasm; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry; D016410:Lymphoma, T-Cell, Cutaneous / Q000097:blood / Q000235:genetics* / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics*; D008928:Mitochondria / Q000737:chemistry; D009363:Neoplasm Proteins / Q000097:blood / Q000235:genetics*; D009928:Organ Specificity; D011132:Polyribosomes / Q000737:chemistry; D012333:RNA, Messenger / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000235:genetics; D012878:Skin Neoplasms / Q000235:genetics / Q000473:pathology; D013347:Subcellular Fractions / Q000737:chemistry; D013382:Subtraction Technique; D013545:Sweat Glands / Q000737:chemistry
D003902:Detergents; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012269:Ribosomal Proteins; D004072:Digitonin