gms | German Medical Science

27. Deutscher Krebskongress

Deutsche Krebsgesellschaft e. V.

22. - 26.03.2006, Berlin

Pylymorphisms of the pKi-67 Promoter and their Biologic Relevance

Meeting Abstract

  • corresponding author presenting/speaker Michael Duchrow - Surgical Research Laboratory, University Clinic of Schleswig-Holstein, Lübeck, Deutschland
  • Daniel Maaser - Surgical Research Laboratory, University Clinic of Schleswig-Holstein, Lübeck
  • Alexandra Wehrstedt - Surgical Research Laboratory, University Clinic of Schleswig-Holstein, Lübeck
  • Hans-Peter Bruch - Surgical Clinic, University Clinic of Schleswig-Holstein, Lübeck
  • Rainer Broll - Surgical Clinic, University Clinic of Schleswig-Holstein, Lübeck

27. Deutscher Krebskongress. Berlin, 22.-26.03.2006. Düsseldorf, Köln: German Medical Science; 2006. DocPO497

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Veröffentlicht: 20. März 2006

© 2006 Duchrow et al.
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Objective: Monoclonal antibodies directed against the nuclear Ki-67 antigen (e.g. MIB-1) are a well-established and widely used marker to access cell proliferation activity, particularly in tumors. The antigen (pKi-67) is solely expressed in actively proliferating cells during all phases of the cell cycle, but it is absent in quiescent cells. PKi-67 is a Ran-associated protein playing an important role in disassembly and reassembly of the nucleoli, depending on the grade of phosphorylation of pKi-67. For colorectal cancer the prognostic relevance of the Ki-67 labeling index is inconsistent. Recently we demonstrated by in situ hybridization, that pKi-67 mRNA expression was principally expressed on a higher level in tumor cells in comparison to normally growing cells. This result may be explained by altered promoter sequences or may be due to differently expressed transcription factors. To investigate whether polymorphisms or mutations of the pKi-67 promoter sequences are responsible for this phenomenon, we sequenced the promoter region of the pKi-67 gene of 50 colorectal cancer patients and 24 healthy donors. To find differences in the biologic relevance of polymorphisms we measured the promoter-activity of all detected variants.

Methods: The pKi-67 promoter region was amplified using Pfu-polymerase (Promega, Mannheim, Germany), randomly cloned into the pCR-Blunt II-Topo-vector (Invitrogen, Groningen, Netherlands), and cycle-sequenced using Thermosequenase (Amersham Pharmacia, Uppsala, Sweden). Firefly-luciferase reporter plasmids were constructed by subcloning of the sequence-controlled inserts into the pGL3-Basic-vector (Promega). The activity of the reporter plasmids was measured using the Dual-Luciferase Reporter Assay (Promega).

Results: After comparison of the cloned promoter sequences we found four polymorphisms (three SNPs and one tetranucleotide repeat) at position –518(A>G), -351(T>C), 186((GGGC)3>(GGGC)5), and –49(G>T) from the transcription start site. We could demonstrate that the variants -186(GGGC)5 (p<0.05), and -49T (p=0.05) were significantly or strongly associated with colorectal cancer. We found the highest promoter activity associated with -186(GGGC)3 and -49T.

Discussion: These results suggest that the two pKi-67 promoter polymorphisms 186(GGGC)3>(GGGC)5 and -49G>T located in the basic promoter may play a crucial rule in the development of colorectal cancer.