Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro.

R115777 [(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. In vitro, using isolated human farnesyl protein transferase, R115777 competitively inhibited the farnesylation of lamin B and K-RasB peptide substrates, with IC50s of 0.86 nM and 7.9 nM, respectively. In a panel of 53 human tumor cell lines tested for growth inhibition, approximately 75% were found to be sensitive to R115777. The majority of sensitive cell lines had a wild-type ras gene. Tumor cell lines bearing H-ras or N-ras mutations were among the most sensitive of the cell lines tested, with responses observed at nanomolar concentrations of R115777. Tumor cell lines bearing mutant K-ras genes required higher concentrations for inhibition of cell growth, with 50% of the cell lines resistant to R115777 up to concentrations of 500 nM. Inhibition of H-Ras, N-Ras, and lamin B protein processing was observed at concentrations of R115777 that inhibited cell proliferation. However, inhibition of K-RasB protein-processing could not be detected. Oral administration b.i.d. of R115777 to nude mice bearing s.c. tumors at doses ranging from 6.25-100 mg/kg inhibited the growth of tumors bearing mutant H-ras, mutant K-ras, and wild-type ras genes. Histological evaluations revealed heterogeneity in tumor responses to R115777. In LoVo human colon tumors, treatment with R115777 produced a prominent antiangiogenic response. In CAPAN-2 human pancreatic tumors, an antiproilferative response predominated, whereas in C32 human melanoma, marked induction of apoptosis was observed. The heterogeneity of histological changes associated with antitumor effects suggested that R115777, and possibly farnesyl protein transferase inhibitors as a class, alter processes of transformation related to tumor-host interactions in addition to inhibiting tumor-cell proliferation.

Characterization of the human N-ras promoter region.

Overexpression of ras proto-oncogenes has been implicated in cancer development. We therefore initiated a study of the human N-ras promoter to determine the regions that control N-ras expression and their potential for interaction with DNA-binding proteins. N-ras CAT constructs were stably integrated into K562 cells by electric field-mediated gene transfer in order to determine functional regions within the human N-ras promoter. A significant proportion of promoter activity was found to lie within a 439 bp fragment comprising an untranslated exon (exon 1) with the adjacent 5' sequence and a small CpG island. A 109 bp [corrected] fragment at the 5' end of exon 1 was essential for promoter activity, while a 45 bp [corrected] deletion from within this region decreased promoter activity by two-thirds. Unlike the human H-ras and mouse K-ras promoters, the N-ras promoter did not exhibit bidirectional activity. DNAse footprinting of the 439 bp fragment revealed seven protected regions, many of which contain sequences homologous to known DNA-binding protein sites (MLTF/myc, CREB/ATF, AP-1, AP-2, myb and E4TF1). In contrast, four putative Sp1 sites did not footprint. Using purified MLTF and appropriate competitors in gel shift and DNAase footprinting assays, we demonstrated binding of MLTF to the MLTF consensus sequence within exon 1.

Allele-specific enrichment: a method for the detection of low level N-ras gene mutations in acute myeloid leukemia.

Point mutations involving codons 12, 13, and 61 of the N-ras gene are found in patients with acute myeloid leukemia (AML). We have developed a sensitive assay for the analysis of these mutations which we have called allele-specific enrichment. In this protocol the polymerase chain reaction (PCR) amplifies DNA with primers that introduce new restriction sites into the normal N-ras allele only. Digestion with the appropriate enzyme cleaves normal, but not mutant, alleles and this digested product provides a mutant allele-enriched template for a second round of amplification. The second PCR product is digested, Southern blotted and analyzed by allele-specific oligonucleotide (ASO) hybridization. This protocol is more sensitive than ASO hybridization alone and has revealed a minor clone in the DNA of a patient with AML. The method may be useful for the detection of minimal residual disease in a subset of patients in remission.

Analysis of expressed N-ras mutations in human melanoma short-term cell lines with allele specific restriction analysis induced by the polymerase chain reaction.

Mutations in ras genes have been found in the DNA of numerous cancer types including melanomas, but the expression of these mutations in melanomas has not yet been addressed. We have used the polymerase chain reaction (PCR) and allele-specific restriction analysis (ASRA) to determine the frequency of expressed N-ras mutations on 25 short-term melanoma tissue culture samples. N-ras cDNA generated using reverse transcriptase from whole cells was used as the PCR template. 14 secondary melanoma cultures that varied in differentiation patterns were analysed. Only 2 were found to express N-ras mutations; in both, the mutation was localised to one of the first two positions of the 61st codon of N-ras. These tumour lines, KMI-M8412a and KMI-M8412b, were established from separate tumour deposits in the same patient. Codons 12 and 13 were found to be free of mutations in all of the lines studied. 8 primary melanomas and 3 unclassified skin lesions were also analysed and found free of N-ras mutations. These results suggest that N-ras may not play such an important role in melanoma tumorigenesis as is speculated by others.

Characterization of K562 cells following introduction of a mutant N-ras gene.

A mutant human N-ras gene (codon 61, C to A substitution) was electroporated into the human leukemic cell line K562, originally derived from a patient with chronic myeloid leukemia (CML) in blast crisis. Despite confirmation of mutant N-ras gene integration and expression, mutant transfected cells exhibited no growth advantage when characterized in suspension cultures and clonogenic assays, and serum deprivation impaired proliferation of both normal and mutant N-ras transfected cells equally. A subclone containing a mutant N-ras gene displayed a proliferation rate and differentiation potential identical to that of non-transfected cells. The failure of N-ras mutations to modify K562 cell behavior is in keeping with the infrequent observation of N-ras mutations in blastic transformation of CML.

Rapid Screening of Mutant N-ras Alleles by Analysis of PCR-Induced Restriction Sites: Allele Specific Restriction Analysis (ASRA).

We have developed a rapid screening method for analysis of codon 12, 13 and 61 N-ras gene mutations, since these mutations have been observed in approximately 25% of patients with acute myeloid leukemia and myelodysplastic syndromes. The method, termed allele specific restriction analysis (ASRA), involves polymerase chain reaction amplification of DNA or RNA using a mismatched primer which introduces appropriately positioned base substitutions in N-ras and creates a restriction site provided the adjacent sequence is normal. Simultaneous analysis of codons 12 and 61 is also possible by the use of a multiprimer reaction mixture. Resistance of the amplified product to digestion indicates the presence of a mutation in the original template. Since ASRA allows simultaneous analysis of mutant and wild type sequences in DNA and RNA, an estimate of the ratio of gene copies and relative expression of N-ras alleles can be obtained for heterozygous individuals.

Analysis of N-ras gene mutations in acute myeloid leukemia by allele specific restriction analysis.

N-ras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of N-ras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant N-ras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. N-ras mutations were more common in patients over 65 years of age (P less than 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of N-ras mutations in leukemia by ASRA and include the first identification of three concurrent N-ras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of N-ras mutations in leukemogenesis.

Detection of K-ras point mutation by enriched PCR-colorimetric plate assay.

Point mutations in the K-ras gene are frequently observed in a variety of human malignancies, including colorectal and pancreatic cancers. In this paper, we describe a sensitive procedure for the detection of point mutations of codon 12 of the K-ras gene. The assay employs a single-tube enriched PCR procedure, coupled to colorimetric detection. In the enriched PCR procedure, the first round of amplification introduces a restriction enzyme site in the wild type, but not in mutant K-ras PCR product. The wild type products are then digested and the second round of PCR enriches for the mutant sequences by amplifying the resistant products. The second round of amplification allows the incorporation of biotin and a substrate binding tag at opposite ends of the mutant product, thus allowing detection of the product by a simple colorimetric assay. The assay has been validated using DNA from a variety of cell lines known to contain either mutant or wild type K-ras. Under these conditions, the assay has proved both reproducible and sensitive, with the ability to detect one mutant molecule in a background of 1000 wild type molecules. The assay allowed discrimination of mutant from wild type K-ras in samples from colonic adenocarcinomas and normal colonic mucosa. The use of a colorimetric detection system reduces observer bias and facilitates analysis of large numbers of samples. As such, the assay may have specific application in the sensitive detection of K-ras mutations in a variety of clinical samples.

Activation of the K-ras oncogene in colorectal neoplasms is associated with decreased apoptosis.

BACKGROUND: Recent in vitro data indicate that the oncogenic effects of activated ras genes may be mediated, at least in part, through inhibition of apoptotic cell death. To examine this proposition in vivo, the relationship between mutations of the K-ras gene and the frequency of apoptosis was studied in a series of 69 sporadic colorectal neoplasms (11 adenomas and 58 carcinomas). METHODS: Mutations in codon 12 of K-ras were determined by a single tube, enriched polymerase chain reaction. Apoptotic cells in tumor sections were identified by in situ end-labeling of fragmented DNA, whereas levels of bcl-2 and p53 proteins were determined by immunohistochemistry. RESULTS: Tumors with mutant K-ras had a significantly lower apoptotic index than those with the wild-type allele (P < 0.05). They were also more likely to exhibit positive bcl-2 staining (P < 0.05). Adenomas showed significantly greater bcl-2 positivity than carcinomas (89% and 51%, respectively; P < 0.05). The frequency of apoptosis in these tumors was not related to either bcl-2 positivity or p53 status. CONCLUSIONS: These findings suggest that activation of K-ras in colorectal carcinoma may inhibit apoptosis and thus favor tumor progression. Alternatively, this association may reflect an accumulation of K-ras mutations in cells in which normal apoptotic pathways have been impaired.

DzyNA-PCR: use of DNAzymes to detect and quantify nucleic acid sequences in a real-time fluorescent format.

BACKGROUND: DzyNA-PCR is a general strategy for the detection and quantification of specific genetic sequences associated with disease or the presence of foreign agents. The method allows homogeneous gene amplification coupled with signal detection in a single closed vessel. METHODS: The strategy involves in vitro amplification of genetic sequences using a DzyNA primer that harbors the complementary (antisense) sequence of a 10-23 DNAzyme. During amplification, amplicons are produced that contain active (sense) copies of DNAzymes that cleave a reporter substrate included in the reaction mixture. The accumulation of amplicons during PCR can be monitored in real time by changes in fluorescence produced by separation of fluoro/quencher dye molecules incorporated into opposite sides of a DNAzyme cleavage site within the reporter substrate. The DNAzyme and reporter substrate sequences can be generic and hence can be adapted for use with primer sets targeting various genes or transcripts. RESULTS: Experiments using K-ras plasmid as template demonstrated that DzyNA-PCR allows quantification of DNA over at least six orders of magnitude (r = 0.992). Studies with human genomic DNA demonstrated the ability to resolve as little as twofold differences in the amount of starting template. DzyNA-PCR allowed the detection of 10 or fewer copies of the target. The clinical utility of the assay was demonstrated using DzyNA-PCR to analyze DNA that was isolated from human serum. CONCLUSION: DzyNA-PCR is a simple, rapid, and sensitive technique for homogeneous amplification and quantification of nucleic acids in clinical specimens.

A rapid PCR ELISA for the detection of activated K-ras in colorectal cancer.

Aims-To develop a rapid PCR ELISA procedure for the detection of mutations in K-ras in a microtitre plate format, and to evaluate the assay for the detection of these mutations in human colorectal cancer.Methods-An enriched PCR method was used with labelled primers, and PCR product was captured on GCN4 coated immunoassay plates. Detection of biotinylated mutant product was performed by colorimetric assay with streptavidin-horseradish peroxidase. The assay was used to determine K-ras status in a series of 60 human colorectal neoplasms, together with paired normal colonic mucosa. Results from gel electrophoretic analysis were compared with ELISA results.Results-The assay proved reliable in detecting K-ras mutations in DNA extracted from both fresh and paraffin embedded colorectal tumours. ELISA results were comparable with results from gel electrophoresis. Mutations of K-ras were detected in 16 of 48 adenocarcinomas and five of 12 adenomas but no mutations were detected in normal mucosa. There was a highly significant difference (p<0.0005) between optical density values for carcinomas with mutant K-ras and their paired normal data. Adenomas did not show the clear distinction between positive and negative results seen with carcinomas.Conclusions-This assay provides a rapid and reliable means of detecting mutations in codon 12 of the K-ras oncogene. The single tube format colorimetric analysis in microtitre plates and clear discrimination between mutant and wild type genes makes the assay suitable for automation. The occurrence of intermediate results in the case of adenomas provides support for the hypothesis that mutations of K-ras occur early in the course of colorectal carcinogenesis.

Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: assay design and optimization.

BACKGROUND: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. METHODS: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. RESULTS: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. CONCLUSION: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.