DNA minor groove binders: Back in the groove

With recent approval of the minor groove binding agent trabectidin in Europe for the treatment of patients with soft tissue sarcomas, there has been renewed interest in minor groove binders. Though previously considered to be without clinical value due to their initial significant toxicities, new minor groove binders are emerging which are challenging that perception. Toxicities in the most recently completed and ongoing trials have been easily manageable. These agents have demonstrable anti-tumor activity against a wide variety of tumor types including leukemias, sarcomas, melanomas, breast and ovarian cancers. Applying these agents according to a particular tumor’s context of vulnerability might reveal previously unconsidered applications for this diverse class of agents. This review provides a look at how minor groove binding agents have progressed from the lab through the clinic with particular emphasis on identifying the contexts of vulnerabilities of patient tumors which increase the effectiveness of these drugs.     

Mismatch repair deficiency is associated with resistance to DNA minor groove alkylating agents.

Mismatch DNA repair deficiency is associated with resistance to certain major groove alkylating agents including methylating agents and cisplatin. We have now studied the relevance of mismatch repair alterations to the cytotoxicity induced by drugs which alkylate N3 adenines in the minor groove of DNA. We have used the mismatch repair defective human colocarcinoma cell line HCT-116 which has a mutation in the hMLH1 gene, and a subline where hMLH1 expression is restored by chromosome 3 transfer (HCT-116+ch3). We have tested three alkylating minor groove binders (tallimustine, carzelesin and CC1065) and one non-covalent minor groove binder (PNU 151807). The HCT-116+ch3 subline was more sensitive than the parental line to the treatment with the three alkylating minor groove binders, while the non-alkylating compound had a similar activity in both cell lines. Further support for mismatch repair being involved in sensitivity of the minor groove alkylators is that two cisplatin-resistant sublines of the human ovarian adenocarcinoma cell line A2780 (A2780/CP70 and A2780/MCP-1) are defective in hMLH1 expression and are more resistant to these agents than the parental mismatch repair proficient cells. Furthermore, the restoration of hMLH1 activity in the A2780/CP70 cell line, by introduction of chromosome 3, was associated with an increased sensitivity to the three alkylating minor groove binders. Again, the non-covalent minor groove binder was equally effective in mismatch repair deficient and proficient clones. The data indicate that mismatch repair deficiency mediated by loss of hMLH1 expression is associated not only with drug-resistance to major groove binders, but also to minor groove binders. However, loss of mismatch repair does not mediate resistance to the non-covalent minor groove binder PNU 151807.     

Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer.

Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma- or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.     

Diaminobenzene schiff base, a novel class of DNA minor groove binder

Molecules that target the deoxyribonucleic acid (DNA) minor groove are relatively sequence specific and they can be excellent carrier structures for cytotoxic chemotherapeutic compounds which can help to minimize side effects. Two novel isomeric derivatives of diaminobenzene Schiff base [N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene (2MJ) and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene (2MH)] were analyzed for their DNA minor groove binding (MGB) ability using viscometry, UV and fluorescence spectroscopy, computational modeling and clonogenic assay. The result shows that 2MJ and 2MH are strong DNA MGBs with the latter being more potent. 2MH can form interstrand hydrogen bond linkages at its oxygens with N3 of adenines. Changing the 2-hydroxy-3-methoxybenzylidene binding position to the 1,3 location on the diaminobenzene structure (2MJ) completely removed any viable hydrogen bond formation with the DNA and caused significant decrease in binding strength and minor groove binding potency. Neither compound showed any significant cytotoxicity towards human breast, colon or liver cancer cell lines.     

Clinical value of bile for the detection of mutant K-ras from colorectal liver metastases

In 25% of patients diagnosed with colorectal cancer, hepatic metastases are not detected at presentation of the colorectal primary but develop during follow-up. Early detection of these metastases may improve the chance of cure by surgical resection. We hypothesised that in patients with occult hepatic metastases, tumour DNA might be detected in bile which could be collected during resection of the colorectal primary. To test this hypothesis, bile from the gall bladder was collected from 17 patients scheduled for resection of evident hepatic metastases (> 2 cm3) from a previously resected colorectal primary. Mutation analysis of the metastases identified five patients (34%) with a K-ras gene mutation in the tumour tissue. These cases were selected for bile analysis for mutant K-ras. Non-mutated DNA could be amplified from all the bile samples, but mutant K-ras could only be detected in bile from one patient. False negative results due to technical deficits could be ruled out by control experiments showing a high DNA isolation efficiency and high sensitivity of the mutation detection method. It is concluded that hepatic metastases, in contrast to pancreatic cancers, do not (regularly) shed mutated DNA into the bile. Hence, molecular screening of bile seems of only limited clinical value for the detection of occult liver metastases.     

Involvement of DNA topoisomerase II in the selective resistance of a mammalian cell mutant to DNA minor groove ligands: ligand-induced DNA-protein crosslinking and responses to topoisomerase poisons

A mutant murine cell line has previously been reported to be resistant to the AT-specific DNA minor groove ligand 2',5'-bi-1H-benzimidazole, 2',(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl), trichloride (Ho33342), due to an enhanced capacity to remove ligand molecules from cellular DNA via a pathway which can be blocked by DNA topoisomerase poisons. We have studied the relationship between ligand resistance and DNA topoisomerase II activity. The cross-sensitivity patterns of the mutant were examined for covalently (anthramycin) and non-covalently (distamycin A) binding minor groove ligands, and DNA intercalating [adriamycin, mitoxantrone and 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA)] and non-intercalating (VP16-213) topoisomerase II poisons. The mutant was cross-resistant to distamycin A alone. The mutant showed no abnormality in: (i) the in vitro decatenation activity of topoisomerase II, (ii) VP16-213 or mAMSA induced protein-DNA cross-linking activities in nuclear extracts, (iii) 'cleavable complex' generation (or DNA strand scisson) in intact cells exposed to topoisomerase poisons. Ho33342 and the topoisomerase II inhibitor novobiocin were found to disrupt both the in vitro binding of nuclear extracted proteins, from mutant and parental cells, to plasmid DNA and the formation of drug-induced cleavable complexes in vitro. Unexpectedly, Ho33342 induced significant levels of DNA-protein crosslinking in both parental and mutant cells. We conclude that: (i) resistance of the mutant is limited to non-covalently binding minor groove ligands, (ii) Ho33342 can block the trapping of DNA topoisomerase II by enzyme poisons in vitro, (iii) Ho33342 can induce a novel form of DNA-protein cross-link in intact cells, and (iv) the resistance of the mutant is not dependent upon some abnormality in topoisomerase II function.     

Rapid and sensitive nonradioactive detection of mutant K-ras genes via 'enriched' PCR amplification.

We have developed a rapid and highly sensitive nonradioactive method for the detection of a mutant codon 12 human c-K-ras allele in the presence of as many as 10(4) copies of the wild type codon 12 allele. This sensitivity is achieved by selective polymerase chain reaction (PCR) amplification of mutant K-ras gene sequences employing a two stage procedure. The first stage entails the amplification of both K-ras mutant and wild type codon 12 sequences, followed by a selective restriction enzyme digestion of only wild type sequences. The second stage involves a subsequent amplification of undigested amplified fragments, enriched in mutant codon 12 sequences. These products are subject to restriction length polymorphism analysis for the detection of point mutations at codon 12. This technique is rapid, nonradioactive, and eliminates the need for either oligonucleotide hybridization or DNA sequencing. Variations of this selective amplification procedure may prove promising for the detection of specific point mutations in heterogenous cell populations.     

Cellular pharmacology of novel C8-linked anthramycin-based sequence-selective DNA minor groove cross-linking agents.

The cellular pharmacology of a series of C8-linked pyrrolobenzodiazepine dimers with polymethylene linkers of n = 3-6 (compounds 1-4) has been studied in a range of human tumour cell lines. The four compounds showed the same pattern of relative activity in five ovarian carcinoma cell lines and one cervical carcinoma cell line with the order of IC50 values of 1 < or = 3 < 4 < 2, which correlated with the previously demonstrated DNA interstrand cross-linking ability of the compounds in plasmid DNA. In human leukaemic K562 cells the agents produced a block in the G2/M phase of the cell cycle characteristic of cross-linking drugs, and extensive interstrand cross-linking was observed in cells by alkaline elution with no evidence of single-strand breaks. Cross-links continued to increase up to 24 h following a 1 h exposure to drug, and no repair was evident by 48 h. In a series of ovarian and cervical carcinoma cell lines with acquired resistance to cisplatin no cross-resistance to the most potent compound 1 was observed in two lines whose major mechanism of resistance to cisplatin was reduced platinum transport. Cross-resistance to 1 was observed in a cell line (A2780cisR) possessing elevated glutathione, and depletion of intracellular glutathione using D,L-buthionine-S,R-sulphoximine (BSO) from 10.25 nmol to 2.8 nmol 10(-6) cells reduced the level of resistance from 11-fold to 2-fold compared with sensitive cells. Cross-linking in the resistant cells was restored to 80% of the level in the parent line by BSO pretreatment. There was also a correlation between glutathione levels and sensitivity to 1 measured in several other ovarian cell lines. Compound 1 also showed cross-resistance in the doxorubicin-resistant cell line 41MdoxR and partial cross-resistance in CH1doxR cells. Both these lines possess elevated levels of p170 glycoprotein. Following treatment with 6 microM verapamil, the resistance in these lines decreased almost 2-fold and 8-fold respectively.     

Control over the sequence specificity of DNA alkylation: syntheses and reactions with 32P-end-labelled DNA of N-alkyl-N-nitrosoureas linked to minor groove binding lexitropsins.

The syntheses of N-2-chloroethyl-N-nitrosoureas (Cl-ENU) that are covalently linked to a series of minor groove binding lexitropsins related to distamycin A are reported. The lexitropsins of 2-Cl-ENU show a sequence specificity for alkylating an adenine toward the ends of its DNA affinity binding domains. The reaction of DNA with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea does not yield these products. Therefore, the linking of the 2-Cl-ENU to the minor groove binder qualitatively and quantitatively alters the DNA observed.     

Isolation of specific DNA probes for detection of Cryptococcus neoformans

Summary. We report the construction of a genomic library and isolation of repetitive DNA probes from Cr. neoformans that are species- and variety-specific. The inserts of the genomic library were in a range of 0.3–1.8 kb, which were in normal distribution in length. Among 592 clones of the genomic library, there were 400(67.6%) clones inserted repetitive DNA sequence and 192(32.4%) inserted single copy DNA sequence of the Cr. neoformans genome. From the library, three specific probes were identified. Clone pCNIIA6 was serotype A-specific, pCNIIB5 species-specific, and pCNIIIG1 varietv-specific (var. neoformans ). On the basis of the high specificity and sensitivity of the signals observed by hybridization, we suggest that these probes provide a means for both biotyping and early diagnosis of Cr. neoformans .
Zusammenfassung. Es wird über den Aufbau einer Genomdatenbank und über die Isolierung repetitiver art- und varietätsspezifischer DNA-Sonden von Cryptococcus neoformans berichtet. Die eingegebenen Fragmente lagen im Bereich von 0.3–1.8 kb. Unter 592 Klonen der Genombank waren 400 (67.6%) Klonfragmente repetitiver DNA und 192 (32.4%) DNA-Einzelkopiefragmente. Aus der Datenbank wurden 3 spezifische Sonden identifiziert. Klon pCNIIA6 war Serotyp-A-spezifisch, pCNIIB5 war artspezifisch, pCNIIIG1 erwies sich als varietätspezifisch var. neoformans ). Im Hinblick auf die hohe Spezifität und Sensitivität der Hybridisierungssignale wird angenommen, daß diese Sonden Hilfsmittel sowohl zur Biotypisierung wie auch zur Schnelldiagnostik von Cr. neoformans eingesetzt werden können.     

荧光定量RT-PCR检测mdr-1基因表达

建立荧光定量ＲＴ－ＰＣＲ检测肿瘤细胞ｍｄｒ－１基因表达的方法,了解肺癌组织中ｍｄｒ的表达水平。方法：建立荧光定量ＲＴ－ＰＣＲ方法,在ＰＥ７７００型检测仪上定量检测Ｋ５６２／ＡＤＭ耐药株和Ｋ５６２不耐药株细胞ｍｄｒ－１有达水平,同时检测４５例初治肺部肿瘤病人组织标本。     

The detection of MDR1 gene expression using fluorogenic probe quantitative RT-PCR method

Competitive RT-PCR and quantative PCR using internal control as reference are usually used to detect the MDR1 gene expression in cancer cells. Both of these methods are end-point detecting method with considerable difference of product amounts between exponential and plateau phase. Therefore it is very difficult to exactly quantify primary template copies. Furthermore, there are some problems with competitive RT-PCR in practice, such as the PCR product contamination that result in fals…     

乳腺癌患者血清游离DNA的定量检测

目的:定量检测乳腺癌患者血清中的游离DNA,并评估其应用前景。方法:采用荧光定量PCR方法检测100例健康女性志愿者、100例乳腺良性疾病患者和200例乳腺癌患者血清游离DNA中GAPDH基因拷贝数,以此作为评估DNA含量的指标。结果:以≥1×103基因拷贝数作为阳性标准,93%健康女性和95%乳腺良性疾病患者DNA检测为阴性,而84.5%乳腺癌患者DNA检测为阳性,其中Ⅰ～Ⅱ期乳腺癌患者阳性率为84%。DNA含量在3组之间的差异有统计学意义(P<0.005)。结论:早期乳腺癌患者血清中存在一定数量肿瘤源性的游离DNA,此类遗传物质可以作为一类特异性的生物学标志物。     

Alpha-bromoacryloyl derivative of distamycin A (PNU 151807): a new non-covalent minor groove DNA binder with antineoplastic activity.

PNU 151807 is a new synthetic alpha-bromoacryloyl derivative of distamycin A. In the present study we investigated the DNA interaction and the mechanism of action of this compound in parallel with the distamycin alkylating derivative, tallimustine. PNU 151807 possesses a good cytotoxic activity in in vitro growing cancer cells, even superior to that found for tallimustine. By footprinting experiments we found that PNU 151807 and tallimustine interact non-covalently with the same AT-rich DNA regions. However, differently from tallimustine, PNU 151807 failed to produce any DNA alkylation as assessed by Taq stop assay and N3 or N7-adenine alkylation assay in different DNA sequences. PNU 151807, like tallimustine, is able to induce an activation of p53, and consequently of p21 and BAX in a human ovarian cancer cell line (A2780) expressing wild-type p53. However, disruption of p53 function by HPV16-E6 does not significantly modify the cytotoxic activity of the compound. Flow cytometric analysis of cells treated with equitoxic concentrations of PNU 151807 and tallimustine showed a similar induction of accumulation of cells in the G2 phase of the cell cycle but with a different time course. When tested against recombinant proteins, only the compound PNU 151807 (and not tallimustine or distamycin A) is able to abolish the in vitro kinase activity of CDK2-cyclin A, CDK2-cyclin E and cdc2-cyclin B complexes. The results obtained showed that PNU 151807 seems to have a mechanism of action completely different from that of its parent compound tallimustine, possibly involving the inhibition of cyclin-dependent kinases activity, and clearly indicate PNU 151807 as a new non-covalent minor groove binder with cytotoxic activity against cancer cells.     

DNA crosslinking, sister chromatid exchange and cytotoxicity of N-2-chloroethylnitrosoureas tethered to minor groove binding peptides

Chloroethylnitrosoureas (CENU) are clinically important chemotherapeuticagents whose mechanism of action involves the formation of interstrandDNA crosslinks via an ethane bridge between N1-G and N3-C. CENUgenerally alkylate G at the N7- and O⁶-positions, with the latterlesion being the precursor to the interstrand crosslink. Inprevious studies, we reported the synthesis of CENU appendedby a C₂H₄ linker to the N-terminus of DNA minor groove bindingdipeptides (lex, information reading peptides) based on N-methylpyrrole-carboxamidesubunits. Because of the dipeptide structure, these CENU-lex'sreact with DNA at adenines associated with lex equilibrium bindingsites. No other CENU has been reported to yield A adducts. Thebiological evaluation of these CENU-lex's show that they aresomewhat less cytotoxic than their simpler counterparts. Inaddition, in vitro studies show that the minor groove bindingCENU-lex's afford a lower level of sister chromatid exchange(SCE) in 9L cells that are sensitive to CENU. There is no differencebetween CENU-lex in SCE induction in 9L-2 cells that are resistantto CENU. Formation of DNA interstrand crosslinks from the CENU-lex'sis lower than for their non-affinity binding analogs in lowionic strength buffer, but similar in the same buffer containing200 mM NaCl. Salt inhibits crosslinking for all CENU, but distamycin,a competitive inhibitor of lex minor groove binding, uniquelyenhances crosslinks for the CENU-lex's. These results are consistentwith the novel minor groove adduction being a ‘detoxification’pathway for the CENU-lex's since this lesion is formed at theexpense of the cytotoxic major groove interstrand crosslink.     

Differential induction of altered gene expression by carcinogens at mutant alleles of a Drosophila locus with a transposable element

Alterations in gene expression by carcinogens were analyzed on three unstable alleles of the white (w+) locus of Drosophilia melanogaster: white-crimson (wc); white-ivory 16 (wi16); and white-unstable 11 (wu11). Two of these alleles (wi16 and wu11) were spontaneous mutant derivatives of wc, which is known to harbor a transposable element. The compounds studied were dimethylnitrosamine, 7,12-dimethylbenz(a)anthracene, and aflatoxin B1. These carcinogens were topically applied on the early larval stages, and the genetic effects assayed were the alterations in eye color either to wild-type (w+) or to other w mutants, initiated both somatically and germinally, as well as the simultaneously induced X-chromosome recessive mutations. The tested compounds influenced the different unstable w alleles in a highly selective manner, both as a function of the inducing agent and the organization of the genome in the target cells. The same treatments raised the somatic reversions to w+ above the corresponding controls for wc and wi16, but not for wu11, whereas the simultaneous induction of other w mutant phenotypes occurred appreciably only with wc. Furthermore, these treatments gave high and variable somatic reversions to w+ with wi16, whereas the simultaneously induced germinal events were uniformly very low. The frequencies of altered expression at the unstable test loci, whether in the soma or germ line, were quantitatively uncorrelated with the mutagenic effects of the treatments in terms of the yield of X-chromosome recessive mutations assayed in the progeny of males emerging from the same treated larvae. There was also an association between the time of the induction of these alterations by the tested carcinogens in the soma and the cellular stage in genomic differentiation. Reversions to w+ were induced preferentially after the onset of genetic determination, whereas changes to the w mutant phenotypes occurred predominantly during the predetermination phases. The genetic properties of transposable elements and the manner of their response to carcinogens supported the hypothesis that nonviral cancer might arise from molecular processes similar to those involved in the evolution of retroviruses.     

Differential expression of the normal and mutated K-ras alleles in chemically induced thymic lymphomas.

The presence of point mutations in the K-ras gene was examined in murine thymic lymphomas induced by a single dose of N-methylnitrosourea by the RNase A mismatch cleavage method and by allelic-specific oligonucleotide hybridization of in vitro amplified DNA by polymerase chain reaction. The results show that the frequency of mutations is lower than that of tumors induced by multiple N-methylnitrosourea treatments. Four mutations identified were the aspartic acid at codon 12, a G:C to A:T transition in its second position. A G:C to T:A transversion in codon 146 was also found in one thymic lymphoma, changing the amino acid alanine to serine. The use of the RNase A assay allowed an estimation of the relative expression levels of both normal and mutant K-ras alleles. The results show that in approximately one half of the tumors the mutant allele is predominantly expressed, suggesting that the normal allele has been lost or that the mutant allele has been amplified relative to the normal. Altogether, these findings are consistent with ras mutations occurring in some instances during tumor development and with a ras effect being not strictly dominant but favoring selection for increasing levels of expression from the oncogenic allele.