Abstract:
:Mutations in the Kirsten ras (KRAS) gene are present in almost all pancreatic adenocarcinomas, and one common mutation is at codon 12: GGT (Gly) is transformed into GAT (Asp). In this work we have targeted the KRAS coding sequence embracing the GAT mutation with a sense PNA molecule (P14), with the aim of downregulating the expression of the mutant allele. P14 was designed with a 15-base sequence complementary to the antisense strand of KRAS at the GAT (Asp) mutation and conjugated to the nuclear localization signal peptide PKKKRKV. CD spectra as a function of temperature show that P14 (2 microM) binds to the antisense strand of the GAT target in the mutated allele with a T(M) of 78 degrees C and to the antisense strand of the GGT target in the wild-type allele with a T(M) of 69 degrees C, in 50 mM Tris-HCl, pH 7.4, and 1 M NaCl. Moreover, P14 showed a high capacity to enter and accumulate in the nuclei of pancreatic cells (Panc-1 and BxPC3), whereas the nonconjugated analogue did not. Quantitative RT-PCR showed that 1 microM P14 was able to specifically suppress KRAS transcription in Panc-1 cells, which harbor mutant KRAS, but not in BxPC3 cells, which contain only wild-type KRAS. However, P14 inhibited KRAS transcription also in BxPC3 cells when used at concentrations of 5 and 10 microM. Following a single PNA treatment, changes in protein level were evident only in Panc-1 cells. As we found that all three genes of the ras family are expressed in the pancreatic cells, we designed PNA-NLS conjugates (P16 and P17) to target also HRAS and NRAS. The binding of each PNA conjugate to the ras genes was assayed by electrophoresis, and their capacity to inhibit transcription was measured by RT-PCR. All of the data obtained, both in vivo and in vitro, are discussed in terms of sequence specificity in the binding between PNA-NLS molecules and genomic DNA.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Cogoi S,Codognotto A,Rapozzi V,Meeuwenoord N,van der Marel G,Xodo LEdoi
10.1021/bi0505215subject
Has Abstractpub_date
2005-08-09 00:00:00pages
10510-9issue
31eissn
0006-2960issn
1520-4995journal_volume
44pub_type
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