Advances in Clinical and Experimental Medicine

Adv Clin Exp Med
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Advances in Clinical and Experimental Medicine

2011, vol. 20, nr 3, May-June, p. 285–294

Publication type: original article

Language: English

Expression of Cell Vault Components MVP, TEP1 and vPARP in Cancerous Ovarian Tissues

Ekspresja składników krypt komórkowych MVP, TEP1 oraz vPARP w tkankach nowotworowych z jajnika

Witold Szaflarski1,, Patrycja Sujka-Kordowska1,, Radosław Januchowski1,, Karolina Jaszczyńska-Nowinka2,, Małgorzata Andrzejewska1,, Adnan Savan3,, Piotr Dzięgiel1,, Piotr Dzięgiel4,, Michał Nowicki1,, Maciej Zabel1,, Maciej Zabel4,

1 Department of Histology and Embryology, Poznań University of Medical Sciences, Poland

2 Department of Gynecological Oncology, Poznań University of Medical Sciences, Poland

3 Department of Biomaterials and Experimental Dentistry, Poznań University of Medical Sciences, Poland

4 Department of Histology and Embryology, Wroclaw Medical University, Poland

Abstract

Background. Cell vaults are composed of three proteins (MVP, TEP1, vPARP) and vRNA. They are known to be involved in the development of resistance mechanisms in cancer cells. There is currently a great deal of controversy over how vaults and their components participate in increasing resistance.
Objectives. The absolute quantification of mRNA molecules for MVP, TEP1 and vPARP genes in normal and cancerous cells, and investigation of the quantitative relations between their levels of expression in cells.
Material and Methods. The study involved 18 surgical specimens from patients diagnosed with ovarian cancer. After RNA isolation from the specimens, the expression of genes encoding MVP, TEP1 and vPARP was determined by quantitative reverse-transcription polymerase chain reaction (RT-QPCR) analysis. In some cases gene expression was confirmed by standard immunohistochemistry. The cancer cells in the specimens were identified by standard H+E staining, correlations with the analyzed gene expression levels were calculated using Spearman’s rank correlation technique and the statistical significance was determined.
Results. The absolute quantification of MVP, TEP1 and vPARP mRNA demonstrated a strong correlation between the expression of MVP and TEP1. To some extent, vPARP acts as a housekeeping gene. In the specimen collection used in the current study, the higher level of MVP expression was not associated with a larger number of ovarian cancer cells.
Conclusion. The RT-QPCR analysis revealed that MVP, TEP1 and vPARP genes are widely expressed in normal and cancerous human tissues; however, MVP and TEP1 might associate with each other in order to perform a still-undefined vault function. The high expression of MVP, TEP1 and vPARP also suggests their ubiquitous role in both normal and malignant cells.

Streszczenie

Wprowadzenie. Krypty komórkowe są zbudowane z 3 białek (MVP, TEP1 i vPARP) oraz vRNA. Struktury te uznaje się za czynniki uczestniczące w rozwoju mechanizmów lekooporności komórek nowotworowych. Dotychczas funkcjonowanie krypt wzbudzało wiele kontrowersji, nie poznano, w jaki sposób krypty oraz ich składniki odpowiadają za zwiększenie odporności.
Cel pracy. Bezwzględne określenie liczby cząsteczek mRNA dla genów MVP, TEP1 oraz vPARP w normalnej i nowotworowej komórce oraz określenie relacji między poziomami ich ekspresji w komórce.
Materiał i metody. Zanalizowano 18 próbek chirurgicznych od pacjentów ze zdiagnozowanym rakiem jajnika. Określono ekspresję genów kodujących MVP, TEP1 oraz vPARP za pomocą ilościowej reakcji PCR w czasie rzeczywistym z zastosowaniem w odwrotnej transkrypcji (RT-QPCR) po wcześniejszym wyizolowaniu RNA z próbek poddanych analizie. Potwierdzenie ekspresji genów w pewnych przypadkach uzyskano za pomocą tradycyjnej immunohistochemii. Przeprowadzono ponadto standardowe barwienie H+E w celu oszacowania liczby komórek nowotworowych w próbkach przeznaczonych do izolacji RNA. Korelacje między zanalizowanymi poziomami ekspresji genów obliczono, stosując korelację rang Spearmana oraz wyznaczając istotność statystyczną dla uzyskanych wyników.
Wyniki. Bezwzględna ocena liczby cząsteczek mRNA dla MVP, TEP1 oraz vPARP wykazała silną korelację między ekspresją MVP i TEP1. vPARP wykazuje pewne cechy genu podstawowego (housekeeping gene). W naszym zbiorze próbek wyższa ekspresja MVP nie jest skorelowana z większą liczbą komórek raka jajnika.
Wnioski. Analiza za pomocą RT-QPCR wykazała, że geny MVP, TEP1 oraz vPARP ulegają szerokiej ekspresji w ludzkich tkankach zmienionych nowotworowo oraz kontrolnych, a białka MVP i TEP1 mogą oddziaływać ze sobą i spełniać nieokreśloną jeszcze funkcję krypty komórkowej. Duża ekspresja MVP, TEP1 oraz vPARP wskazuje również na ich powszechną rolę w każdej komórce, normalnej i zmienionej nowotworowo.

Key words

cancer, cell vaults, MVP/LRP, TEP1, vPARP

Słowa kluczowe

nowotwory, krypty komórkowe, MVP/LRP, TEP1, vPARP

References (35)

  1. Kedersha NL, Rome LH: Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA. J Cell Biol 1986, 103, 699–709.
  2. Rome L, Kedersha N, Chugani D: Unlocking vaults: organelles in search of a function. Trends Cell Biol 1991, 1, 47–50.
  3. Kickhoefer VA, Siva AC, Kedersha NL, Inman EM, Ruland C, Streuli M, Rome LH: The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J Cell Biol 1999, 146, 917–928.
  4. Kickhoefer VA, Stephen AG, Harrington L, Robinson MO, Rome LH: Vaults and telomerase share a common subunit, TEP1. J Biol Chem 1999, 274, 32712–32717.
  5. Kickhoefer VA, Searles RP, Kedersha NL, Garber ME, Johnson DL, Rome LH: Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III. J Biol Chem 1993, 268, 7868–7873.
  6. Kedersha NL, Miquel MC, Bittner D, Rome LH: Vaults. II. Ribonucleoprotein structures are highly conserved among higher and lower eukaryotes. J Cell Biol 1990, 110, 895–901.
  7. Vasu SK, Kedersha NL, Rome LH: cDNA cloning and disruption of the major vault protein alpha gene (mvpA) in Dictyostelium discoideum. J Biol Chem 1993, 268, 15356–15360.
  8. Vasu SK, Rome LH: Dictyostelium vaults: disruption of the major proteins reveals growth and morphological defects and uncovers a new associated protein. J Biol Chem 1995, 270, 16588–16594.
  9. Herrmann C, Zimmermann H, Volknandt W: Analysis of a cDNA encoding the major vault protein from the electric ray Discopyge ommata. Gene 1997, 188, 85–90.
  10. Vollmar F, Hacker C, Zahedi RP, Sickmann A, Ewald A, Scheer U, Dabauvalle MC: Assembly of nuclear pore complexes mediated by major vault protein. J Cell Sci 2009, 122, 780–786.
  11. Kedersha NL, Heuser JE, Chugani DC, Rome LH: Vaults. III. Vault ribonucleoprotein particles open into flower-like structures with octagonal symmetry. J Cell Biol 1991, 112, 225–235.
  12. Stephen AG, Raval-Fernandes S, Huynh T, Torres M, Kickhoefer VA, Rome LH: Assembly of vault-like particles in insect cells expressing only the major vault protein. J Biol Chem 2001, 276, 23217–23220.
  13. van Zon A, Mossink MH, Schoester M, Scheffer GL, Scheper RJ, Sonneveld P, Wiemer EA: Structural domains of vault proteins: a role for the coiled coil domain in vault assembly. Biochem Biophys Res Commun 2002, 291, 535–541.
  14. de Murcia G, Menissier-de Murcia J, Schreiber V: Poly(ADP-ribose) polymerase: molecular biological aspects. Bioessays 1991, 13, 455–462.
  15. de Murcia JM, Niedergang C, Trucco C, Ricoul M, Dutrillaux B, Mark M, Oliver FJ, Masson M, Dierich A, LeMeur M, Walztinger C, Chambon P, de Murcia G: Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells. Proc Natl Acad Sci U S A 1997, 94, 7303–7307.
  16. Harrington L, McPhail T, Mar V, Zhou W, Oulton R, Bass MB, Arruda I, Robinson MO: A mammalian telomeraseassociated protein. Science 1997, 275, 973–977.
  17. Mikyas Y, Makabi M, Raval-Fernandes S, Harrington L, Kickhoefer VA, Rome LH, Stewart PL: Cryoelectron microscopy imaging of recombinant and tissue derived vaults: localization of the MVP N termini and VPARP. J Mol Biol 2004, 344, 91–105.
  18. Tanaka H, Kato K, Yamashita E, Sumizawa T, Zhou Y, Yao M, Iwasaki K, Yoshimura M, Tsukihara T: The structure of rat liver vault at 3.5 angstrom resolution. Science 2009, 323, 384–388.
  19. Weinrich SL, Pruzan R, Ma L, Ouellette M, Tesmer VM, Holt SE, Bodnar AG, Lichtsteiner S, Kim NW, Trager JB, Taylor RD, Carlos R, Andrews WH, Wright WE, Shay JW, Harley CB, Morin GB: Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT. Nat Genet 1997, 17, 498–502.
  20. Scheffer GL, Wijngaard PL, Flens MJ, Izquierdo MA, Slovak ML, Pinedo HM, Meijer CJ, Clevers HC, Scheper RJ: The drug resistance-related protein LRP is the human major vault protein. Nat Med 1995, 1, 578–582.
  21. Izquierdo MA, Scheffer GL, Flens MJ, Giaccone G, Broxterman HJ, Meijer CJ, van der Valk P, Scheper RJ: Broad distribution of the multidrug resistance-related vault lung resistance protein in normal human tissues and tumors. Am J Pathol 1996, 148, 877–887.
  22. Kickhoefer VA, Rajavel KS, Scheffer GL, Dalton WS, Scheper RJ, Rome LH: Vaults are up-regulated in multidrugresistant cancer cell lines. J Biol Chem 1998, 273, 8971–8974.
  23. Kitazono M, Okumura H, Ikeda R, Sumizawa T, Furukawa T, Nagayama S, Seto K, Aikou T, Akiyama S: Reversal of LRP-associated drug resistance in colon carcinoma SW-620 cells. Int J Cancer 2001, 91, 126–131.
  24. Kitazono M, Sumizawa T, Takebayashi Y, Chen ZS, Furukawa T, Nagayama S, Tani A, Takao S, Aikou T, Akiyama S: Multidrug resistance and the lung resistance-related protein in human colon carcinoma SW-620 cells. J Natl Cancer Inst 1999, 91, 1647–1653.
  25. Siva AC, Raval-Fernandes S, Stephen AG, LaFemina MJ, Scheper RJ, Kickhoefer VA, Rome LH: Up-regulation of vaults may be necessary but not sufficient for multidrug resistance. Int J Cancer 2001, 92, 195–202.
  26. Huffman KE, Corey DR: Major vault protein does not play a role in chemoresistance or drug localization in a nonsmall cell lung cancer cell line. Biochemistry 2005, 44, 2253–2261.
  27. Mossink MH, van Zon A, Franzel-Luiten E, Schoester M, Kickhoefer VA, Scheffer GL, Scheper RJ, Sonneveld P, Wiemer EA: Disruption of the murine major vault protein (MVP/LRP) gene does not induce hypersensitivity to cytostatics. Cancer Res 2002, 62, 7298–7304.
  28. van Zon A, Mossink MH, Schoester M, Scheper RJ, Sonneveld P, Wiemer EA: Efflux kinetics and intracellular distribution of daunorubicin are not affected by major vault protein/lung resistance-related protein (vault) expression. Cancer Res 2004, 64, 4887–4892.
  29. Lee C, Lee S, Shin SG, Hwang S: Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli. Appl Microbiol Biotechnol 2008, 78, 371–376.
  30. Holschneider CH, Berek JS: Ovarian cancer: epidemiology, biology, and prognostic factors. Semin Surg Oncol 2000, 19, 3–10.
  31. Szakacs G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM: Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006, 5, 219–234.
  32. Meijer GA, Schroeijers AB, Flens MJ, Meuwissen SG, van der Valk P, Baak JP, Scheper RJ: Increased expression of multidrug resistance related proteins Pgp, MRP1, and LRP/MVP occurs early in colorectal carcinogenesis. J Clin Pathol 1999, 52, 450–454.
  33. Bouhamyia L, Chantot-Bastaraud S, Zaidi S, Roynard P, Prengel C, Bernaudin JF, Fleury-Feith J: Immunolocalization and cell expression of lung resistance-related protein (LRP) in normal and tumoral human respiratory cells. J Histochem Cytochem 2007, 55, 773–782.
  34. Chugani DC, Rome LH, Kedersha NL: Evidence that vault ribonucleoprotein particles localize to the nuclear pore complex. J Cell Sci 1993, 106, 23–29.
  35. Dickenson NE, Moore D, Suprenant KA, Dunn RC: Vault ribonucleoprotein particles and the central mass of the nuclear pore complex. Photochem Photobiol 2007, 83, 686–691.