Clinical & Experimental Metastasis

, Volume 19, Issue 7, pp 571–582

Quantification of human Alu sequences by real-time PCR – an improved method to measure therapeutic efficacy of anti-metastatic drugs in human xenotransplants

  • Tanja Schneider
  • Franz Osl
  • Thomas Friess
  • Hubertus Stockinger
  • Werner V. Scheuer


For measuring the efficacy of new anti-metastatic drugs in preclinical models, macroscopical analysis or classical histology of secondary organs are established methods. However, macroscopical evaluation does not take into consideration intra-organ metastasis. Histological analysis is often performed in few sections of the relevant organs, and this may be misleading, since equal distribution of tumor cells within an organ is unlikely. In addition, recent studies have demonstrated that anti-tumorigenic drugs are able to promote metastasis and to change the metastatic pattern. Therefore, extensive analysis of metastasis is mandatory for the evaluation of new compounds. A feasibility study was conducted to find out if the quantification of human Alu sequences could be applied as a surrogate marker for metastasis in xenografts. Alu PCR was performed by using the LightCycler® system,* which allows PCR reaction and subsequent quantification of the PCR products in less than 30 min. We found that i) the equivalent of one human tumor cell in 1 × 106 murine cells could be detected; ii) in tumor-carrying mice, Alu signal increased over time in secondary organs; iii) this increase was more prominent using highly metastatic tumor cells; iv) Alu signal intensity in DNA extracted from tissue slides correlated with the expression of histological tumor markers; v) in three different tumor models (colon, breast and lung), treatment with Taxol or 5-fluorouracil reduced the amount of Alu in different organs. In contrast, reduction of Alu by the matrix metalloproteinase inhibitor RO 28-2653 was not significant. Taken together, quantification of Alu sequences is a fast and accurate method to evaluate the therapeutic efficacy of anti-metastatic drugs in xenografts.

Alu sequences LightCycler® system metastasis PCR real-time PCR xenografts 


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  1. 1.
    Dimitroff CJ, Sharma A, Bernacki RJ. Cancer metastasis: A search for therapeutic inhibition. Cancer Invest 1998; 16(4): 279–90.PubMedGoogle Scholar
  2. 2.
    Zetter BR. Angiogenesis and tumor metastasis. Annu Rev Med 1998; 49: 407–24.PubMedCrossRefGoogle Scholar
  3. 3.
    Kerbel RS, Viloria-Petit A, Klement G et al. 'Accidental’ anti-angiogenic drugs: Anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples. Eur J Cancer 2000; 36(10): 1248–57.PubMedCrossRefGoogle Scholar
  4. 4.
    Garofalo A, Chirivi RGS, Scanziani E et al. Comparative study on the metastatic behavior of human tumors in nude, beige/nude/xid and severe combined immunodeficient mice. Invasion Metastasis 1993; 13(2): 82–91.PubMedGoogle Scholar
  5. 5.
    Friess T, Krell HW, Klaus J et al. Ro 28-2653, a new MMP inhibitor suppresses primary tumor growth and metastatic spread of human lung carcinoma H460M2 in SCID beige mice. VIII International Congress of the Metastasis Research Society, London, UK, 2000. Clin Exp Metastasis 1999; 17(9): 771.Google Scholar
  6. 6.
    Jojovic M, Schumacher U. Quantitative assessment of spontaneous lung metastases of human HT29 colon cancer cells transplanted into SCID mice. Cancer Lett 2000; 152(2): 151–6.PubMedCrossRefGoogle Scholar
  7. 7.
    Blaheta HJ, Sotlar K, Breuninger H et al. Does intensive histopathological workup by serial sectioning increase the detection of lymph node micrometastasis in patients with primary cutaneous melanoma? Melanoma Res 2001; 11(1): 57–63.PubMedCrossRefGoogle Scholar
  8. 8.
    Waye JS, Presley LA, Budowle B et al. A simple and sensitive method for quantifying human genomic DNA in forensic specimen extracts. Biotechniques 1989; 7(8): 852–5.PubMedGoogle Scholar
  9. 9.
    McKenzie BA, Barrieux A, Varki NM. A novel detection system for submicroscopic human metastasis in athymic mice. Cancer Commun 1991; 3(1): 15–9.PubMedGoogle Scholar
  10. 10.
    Shoemaker RH, Smythe AM, Wu L et al. Evaluation of metastatic human tumor burden and response therapy in a nude mouse xenograft model using a molecular probe for repetitive human DNA sequences. Cancer Res 1992; 52(10): 2791–6.PubMedGoogle Scholar
  11. 11.
    Weisberg TF, Cahill BK, Vary CPH. Non-radioisotopic detection of human xenogeneic DNA in a mouse transplantation model. Mol Cell Probes 1996; 10(2): 139–46.PubMedCrossRefGoogle Scholar
  12. 12.
    Zubair AC, Ali SA, Rees RC et al. Investigation of the effect of BB-94 (batimastat) on the colonization potential of human lymphoma cells in SCID mice. Cancer Lett 1996; 107(1): 91–5.PubMedCrossRefGoogle Scholar
  13. 13.
    Kim J, Yu W, Kovalski K et al. Requirement for specific proteases in cancer cell intravasation as revealed by a novel semiquantitative PCR-based assay. Cell 1998; 94(3): 353–62.PubMedCrossRefGoogle Scholar
  14. 14.
    Wittwer CT, Ririe KM, Andrew RV et al. The LightCycler®: A microvolume multisample fluorimeter with rapid temperature control. BioTechniques 1997; 22(1): 176–81.PubMedGoogle Scholar
  15. 15.
    Corti C, Pratesi G, De Cesare M et al. Spontaneous lung metastases in a human lung tumor xenograft: A new experimental model. J. Cancer Res Clin Oncol 1996; 122(3): 154–60.PubMedCrossRefGoogle Scholar
  16. 16.
    Uchida J, Sato K, Okabe H et al. Experimental postoperative adjuvant chemotherapy by UFT using primary tumor amputation model. Int J Mol Med 2000; 5(4): 357–62.PubMedGoogle Scholar
  17. 17.
    Bagheri-Yarmand R, Kourbali Y, Rath AM et al. Carboxymethyl benzylamide dextran blocks angiogenesis of MDA-MB435 breast carcinoma xenografted in fat pad and its lung metastases in nude mice. Cancer Res 1999; 59(3): 507–10.PubMedGoogle Scholar
  18. 18.
    Alves F, Borchers U, Padge B et al. Inhibitory effect of a matrix metalloproteinase inhibitor on growth and spread of human pancreatic adenocarcinima evaluated in an orthotopic severe combined immunodeficient (SCID) mouse model. Cancer Lett 2001; 165(2): 161–70.PubMedCrossRefGoogle Scholar
  19. 19.
    Price JE, Polyzos A, Zhang RD et al. Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 1990; 50(3): 717–21.PubMedGoogle Scholar
  20. 20.
    Sharma R, Adam E, Schumacher U. The action of 5-fluorouracil on human HT29 colon cancer cells grown in SCID mice: Mitosis, apoptosis and cell differentiation. Br J Cancer 1997; 76(8): 1011–6.PubMedGoogle Scholar
  21. 21.
    Kariya Y, Kato K, Hayashizaki Y et al. Revision of consensus sequence of human Alu repeats — a review. Gene 1987; 53(1): 1–10.PubMedCrossRefGoogle Scholar
  22. 22.
    Arcot SS, Shaikh TH, Kim J et al. Sequence diversity and chromosomal distribution of ‘young’ Alu repeats. Gene 1995; 163(2): 273–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Geldof AA, Rao BR. Doxorubicin treatment increases metastasis of prostate tumor (R3327-MatLyLu). Anticancer Res 1988; 8(6): 1335–40.PubMedGoogle Scholar
  24. 24.
    Murphy SB. Secondary acute myeloid leukemia following treatment with epipodophyllotoxins. J Clin Oncol 1993; 11(2): 199–201.PubMedGoogle Scholar
  25. 25.
    De Larco JE, Wuertz BRK, Manivel C et al. Progression and enhancement of metastatic potential after exposure of tumor cells to chemotherapeutic agents. Cancer Res 2001; 61(7): 2857–61.PubMedGoogle Scholar
  26. 26.
    Krueger A, Soeltl R, Sopov I et al. Hydroxamate-type matrix metalloproteinase inhibitor Baltimastat promotes liver metastasis. Cancer Res 2001; 61(4): 1272–5.Google Scholar
  27. 27.
    Cameron MD, Schmidt EE, Kerkvliet N et al. Temporal progression of metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Res 2000; 60(5): 2541–6.PubMedGoogle Scholar
  28. 28.
    Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/ type IV collagenase (MMP-9). J Biol Chem 1997; 272(46): 28823–5.PubMedCrossRefGoogle Scholar
  29. 29.
    O'Reilly MS, Wiederschain D, Stetler-Stevenson WG. Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance. J Biol Chem 1999; 274(41): 29568–71.PubMedCrossRefGoogle Scholar
  30. 30.
    Oba K, Konno H, Tanaka T et al. Prevention of liver metastasis of human colon cancer by selective matrix metalloproteinase inhibitor MMI-166. Cancer Lett 2002; 175: 45–51.PubMedCrossRefGoogle Scholar
  31. 31.
    Matsushita A, Onda M, Uchida E et al. Antitumor effect of a new selective matrix metalloproteinase inhibitor, MMI-166, on experimental pancreatic cancer. Int J Cancer 2001; 92: 434–40.PubMedCrossRefGoogle Scholar
  32. 32.
    Yang M, Baranov E, Jiang P et al. Whole body optical imaging of green fluorescent protein-expressing tumors and metastases. Proc Natl Acad Sci 2000; 97(3): 1206–11.PubMedCrossRefGoogle Scholar
  33. 33.
    Kruger A, Schirrmacher V, Khokha R. The bacterial lacZ gene: An important tool for metastasis research and evaluation of new cancer therapies. Cancer Metastasis Rev 1998–1999; 17(3): 285–94.PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang L, Hellstrom KE, Chen L. Luciferase activity as a marker of tumor burden and as an indicator of tumor response to antineoplastic therapy in vivo. Clin Exp Metastasis 1994; 12(2): 87–92.PubMedCrossRefGoogle Scholar
  35. 35.
    Migliaccio AR, Bengra C, Ling J et al. Stable and unstable transgene integration sites in the human genome: Extinction of the green fluorescent protein transgene in K562 cells. Gene 2000; 256(1–2): 197–214.PubMedCrossRefGoogle Scholar
  36. 36.
    Coralli C, Cemazar M, Kanthou C et al. Limitations of the reporter green fluorescent protein under simulated tumor conditions. Cancer Res 2001; 61(12): 4784–90.PubMedGoogle Scholar
  37. 37.
    Lojda Z. Indigogenic methods for glycosidases. II. An improved method for beta-D-galactosidase and its application to localization studies of the enzymes in intestine and in other tissues. Histochemie 1970; 23(3): 266–88.PubMedCrossRefGoogle Scholar
  38. 38.
    Contag CH, Jenkins D, Contag PR et al. Use of reporter genes for optical measurements of neoplastic disease in vivo. Neoplasia 2000; 2(1–2): 41–52.PubMedCrossRefGoogle Scholar
  39. 39.
    Ren Y, Savill J. Apoptosis: the importance of being eaten. Cell Death Different 1998; 5(7): 563–8.CrossRefGoogle Scholar
  40. 40.
    Enari M, Sakahira H, Yokoyama H et al. A caspase-activated Dnase that degrades DNA during apoptosis and its inhibitor ICAD. Nature 1998; 391(6662): 43–50.PubMedCrossRefGoogle Scholar
  41. 41.
    Okada C, Mizuochi T. Role of macrophage lysosomal enzymes in the degradation of nucleosomes of apoptotic cells. J Immunol 1999; 163(10): 5346–52.Google Scholar
  42. 42.
    McIlroy D, Tanaka M, Sakahira H et al. An auxiliary mode of apoptotic DNA fragmentation provided by macrophages. Genes Dev 2000; 14(5): 549–58.PubMedGoogle Scholar
  43. 43.
    Urban C, Gruber F, Kundi M et al. A systematic and quantitative analysis of PCR template contamination. J Forensic Sci 2000; 45(6): 1307–11.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Tanja Schneider
    • 1
  • Franz Osl
    • 1
  • Thomas Friess
    • 1
  • Hubertus Stockinger
    • 2
  • Werner V. Scheuer
    • 1
  1. 1.Departments of Molecular PharmacologyRoche Diagnostics GmbHPenzbergGermany
  2. 2.Applied ScienceRoche Diagnostics GmbHPenzbergGermany

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