Activity of MGCD290, a Hos2 histone deacetylase inhibitor, in combination with azole antifungals against opportunistic fungal pathogens.

We report on the in vitro activity of the Hos2 fungal histone deacetylase (HDAC) inhibitor MGCD290 (MethylGene, Inc.) in combination with azoles against azole-resistant yeasts and molds. Susceptibility testing was performed by the CLSI M27-A3 and M38-A2 broth microdilution methods. Testing of the combinations (MGCD290 in combination with fluconazole, posaconazole, or voriconazole) was performed by the checkerboard method. The fractional inhibitory concentrations were determined and were defined as <0.5 for synergy, >or=0.5 but <4 for indifference, and >or=4 for antagonism. Ninety-one isolates were tested, as follows: 30 Candida isolates, 10 Aspergillus isolates, 15 isolates of the Zygomycetes order, 10 Cryptococcus neoformans isolates, 8 Rhodotorula isolates, 8 Fusarium isolates, 5 Trichosporon isolates, and 5 Scedosporium isolates. MGCD290 showed modest activity when it was used alone (MICs, 1 to 8 microg/ml) and was mostly active against azole-resistant yeasts, but the MICs against molds were high (16 to >32 microg/ml). MGCD290 was synergistic with fluconazole against 55 (60%) of the 91 isolates, with posaconazole against 46 (51%) of the 91 isolates, and with voriconazole against 48 (53%) of the 91 isolates. Synergy between fluconazole and MGCD290 was observed against 26/30 (87%) Candida isolates. All 23 of the 91 Candida isolates that were not fluconazole susceptible demonstrated a reduced fluconazole MIC that crossed an interpretive breakpoint (e.g., resistant [MIC, >or=64 microg/ml] to susceptible [MIC,

A phase I pharmacokinetic and pharmacodynamic study of the DNA methyltransferase 1 inhibitor MG98 administered twice weekly.

BACKGROUND: Hypermethylation and inactivation of tumor suppressor genes by the enzyme DNA methyltransferase may lead to neoplastic transformation. MG98, a phosphorothioate antisense oligodeoxynucleotide that is a specific inhibitor of mRNA for human DNA methyltransferase 1 (DNMT1), was evaluated in a phase I study. PATIENTS AND METHODS: MG98 was given as a 2 h i.v. infusion twice weekly three weeks out of every four to patients with solid tumors. Pharmacokinetic evaluation was performed on days 1 and 15 of cycle 1 and mRNA expression of DNMT1 was measured in peripheral blood mononuclear cells (PBMCs). RESULTS: Nineteen patients were entered onto the study. A total of 74 cycles (range 1-18 cycles) were administered at dose levels from 40 to 480 mg/m(2). Dose limiting toxicity was seen in two of three patients at 480 mg/m(2) and consisted of a constellation of fever, chills, fatigue and, in one case, confusion beginning within 6 h after the first infusion. Other toxic effects included fatigue, anorexia, nausea, vomiting and diarrhea, reversible elevations in transaminases and partial thromboplastin time. Pharmacokinetic evaluation showed C(max) and AUC to be dose proportional with low inter- and intra-patient variability. No consistent changes in DNMT1 mRNA expression were noted in PBMCs. One partial response was documented in a patient with renal cell carcinoma treated at 80 mg/m(2). CONCLUSIONS: The recommended dose of MG98 was 360 mg/m(2) given by 2 h infusion twice a week for three weeks out of every four. Phase II trials using this dose and schedule are underway.

Modulation of the multidrug-resistance phenotype by new tropane alkaloid aromatic esters from Erythroxylum pervillei.

Nine tropane alkaloid aromatic esters (1-9) were isolated from the roots of Erythroxylum pervillei by following their potential to reverse multidrug-resistance with vinblastine-resistant oral epidermoid carcinoma (KB-V1) cells. All isolates, including seven new structures (3-9), were evaluated against a panel of human cancer cell lines, and it was found that alkaloids 3 and 5-9 showed the greatest activity with KB-V1 cells assessed in the presence of vinblastine, suggesting that these new compounds are potent modulators of P-glycoprotein. Confirmatory results were obtained with human ovarian adenocarcinoma (SKVLB) cells evaluated in the presence of adriamycin and synergistic studies performed with several cell lines from the NCI tumor panel. The structures of the new compounds were determined using spectroscopic techniques. Single-crystal X-ray analysis was performed on the monoester, tropane-3 alpha,6 beta,7 beta-triol 3-phenylacetate (1).

Down-regulation of human DNA-(cytosine-5) methyltransferase induces cell cycle regulators p16(ink4A) and p21(WAF/Cip1) by distinct mechanisms.

A common event in the development of human neoplasia is the loss of growth regulatory tumor suppressor functions. Methylation of 5' CpG islands of tumor suppressor genes and elevated levels of the DNA-(cytosine-5)-methyltransferase enzyme (DNA MeTase) are also prevalent features of human neoplasia. However, direct evidence that elevated DNA MeTase levels alter gene expression and influence oncogenesis has been difficult to obtain, in part due to the lack of specific DNA MeTase inhibitors. Here we show that specific reduction of cellular DNA MeTase levels in human cancer cells with potent antisense inhibitors: 1) causes demethylation of the p16(ink4A) gene promoter; 2) causes re-expression of the p16(ink4A) protein; 3) leads to accumulation of the hypophosphorylated form of the retinoblastoma protein (pRb); and 4) inhibits cell proliferation. Stepwise reduction of cellular DNA MeTase protein levels also induced a corresponding rapid increase in the cell cycle regulator p21(WAF/Cip1) protein demonstrating a regulatory link between DNA MeTase and the growth regulator p21(WAF/Cip1) that is independent of methylation of DNA. These results suggest that the elevated levels of DNA MeTase seen in cancer cells can inhibit tumor suppressors by distinct mechanisms involving either transcriptional inactivation through DNA methylation or by a methylation independent regulation.

Topoisomerase I inhibition by the camptothecin analog Gl147211C. From the laboratory to the clinic.

The development of camptothecin-like compounds as inhibitors of DNA topoisomerase I for the treatment of solid tumors has generated clinical excitement in this new class of drugs. We have discovered, developed, and entered into clinical trial a novel, potent, and water-soluble camptothecin analog with significant antitumor activity. This compound, Gl147211C [7-(4-methylpiperaziinomethylene)-10, 11-ethylenedioxy-20(S)-camptothecin hydrochloride] is a specific inhibitor of DNA topoisomerase I. Compared to topotecan, Gl147211C is approximately three times as potent in the cleavable complex assay and approximately twice as soluble in aqueous medium. Human tumor cell line cytotoxicity assays indicated that Gl147211C was approximately 3- to 5-fold more potent than topotecan, while both compounds were relatively insensitive to the multidrug resistance P-glycoprotein. The in vivo preclinical antitumor activity of Gl147211C was compared to topotecan in an array of human tumor xenograft models in nude mice. In general, Gl147211C was able to induce regression of established tumors whereas topotecan was not. Microscopic evaluation of necropsied tissues indicated that drug-induced toxicity was mild, primarily limited to the gastrointestinal tract, and was comparable for both Gl147211C and topotecan. Based on these observations, Gl147211C moved through preclinical development and subsequently into Phase I clinical trial. A summary of Phase I trial results to date is provided.

Lupane derivatives from Lophopetalum wallichii with farnesyl protein transferase inhibitory activity.

Chloroform-soluble extracts of the stems and of the mixed stems and stem bark of Lophopetalum wallichii were found to be inhibitory in a farnesyl protein transferase (FPTase) bioassay system. During the course of activity-guided fractionation, the known lupane-type triterpenes, ochraceolide A (1), ochraceolide B (2), betulin, and lupeol and the new lupane lactone, dihydro ochraceolide A (4), were isolated. The stereochemistry of the epoxide group of ochraceolide B (2) was determined by preparation of both epoxide isomers [2, and the new semisynthetic derivative, 20-epi-ochraceolide B (3)] from 1. The structure of 4 was established by reduction of 1 with sodium borohydride. Compounds 1 and 2 exhibited significant inhibitory activity in the FPTase assay (IC50 values of 1.0 and 0.7 microgram/mL, respectively). Lupeol was found to be weakly active (IC50 65.0 micrograms/mL) in this test system, whereas no significant inhibition was detected for betulin or compounds 3 or 4. When evaluated against a panel of human cancer cells in culture, compounds 1 and 4 were modestly cytotoxic. Compounds 2 and 3 were not active in the panel.

Natural-product inhibitors of human DNA ligase I.

Enzymatic activity mediated by recombinant human DNA ligase I (hLI), in conjunction with tannin removal procedures, has been applied to a natural-product screen involving approximately 1000 plant extracts and various pure compounds. The primary hLI activity assay involved the measurement of the amount of radiolabelled phosphate in a synthetic nucleic acid hybrid that becomes resistant to alkaline phosphatase as a result of ligation. A bioactivity-guided fractionation scheme resulted in the isolation of ursolic [IC50=100 micrograms/ml (216 microM)] and oleanolic [IC50=100 micrograms/ml (216 microM)] acids from Tricalysia niamniamensis Hiern (Rubiaceae), which demonstrated similar DNA ligase inhibition profiles to other triterpenes such as aleuritolic acid. Protolichesterinic acid [IC50=6 micrograms/ml (20 microM)], swertifrancheside [IC50 = 8 micrograms/ml(11)microM)] and fulvoplumierin [IC50=87 micrograms/ml (357 microM)] represent three additional natural-product structural classes that inhibit hLI. Fagaronine chloride [IC50=10 micrograms/ml (27 micronM] and certain flavonoids are also among the pure natural products that were found to disrupt the activity of the enzyme, consistent with their nucleic acid intercalative properties. Further analyses revealed that some of the hLI-inhibitory compounds interfered with the initial adenylation step of the ligation reaction, indicating a direct interaction with the enzyme protein. However, in all cases, this enzyme-inhibitor interaction did not disrupt the DNA relaxation activity mediated by hLI. These results indicate that, although the same enzyme active site may be involved in both enzyme adenylation and DNA relaxation, inhibitors may exert allosteric effects by inducing conformational changes that disrupt only one of these activities. Studies with inhibitors are important for the assignment of specific cellular functions to these enzymes, as well as for their development into clinically useful antitumour agents.

Water soluble inhibitors of topoisomerase I: quaternary salt derivatives of camptothecin.

Eleven water soluble 7-substituted quaternary ammonium salt derivatives of 10,11-(methylenedioxy)- and 10,11-(ethylenedioxy)-(20S)-camptothecin were synthesized via the Friedlander reaction followed by nucleophilic displacement with an aromatic amine. All of these compounds were more potent than camptothecin in the in vitro cleavable complex assay. These inherently charged camptothecin derivatives were cytotoxic against three different human tumor cell lines (SKOV3, an ovarian adenocarcinoma; SKVLB a multidrug resistant ovarian adenocarcinoma; and HT-29, a colon carcinoma). A selected group of five compounds was evaluated in the nude mouse HT-29 xenograft model. Two of these quaternary salts (17 and 18) were more efficacious than Topotecan in delaying tumor growth. In an extended in vivo model, 18 demonstrated tumor regression.

Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia.

Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15-oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines. The structures of these compounds were determined by analysis of their spectroscopic data. The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition. The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol. The adduct with beta-mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself. The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677. The effects of 13-methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells. Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed. The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific. No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15-oxozoapatlin. These data indicate that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression.

Mammalian DNA ligase III: molecular cloning, chromosomal localization, and expression in spermatocytes undergoing meiotic recombination.

Three biochemically distinct DNA ligase activities have been identified in mammalian cell extracts. We have recently purified DNA ligase II and DNA ligase III to near homogeneity from bovine liver and testis tissue, respectively. Amino acid sequencing studies indicated that these enzymes are encoded by the same gene. In the present study, human and murine cDNA clones encoding DNA ligase III were isolated with probes based on the peptide sequences. The human DNA ligase III cDNA encodes a polypeptide of 862 amino acids, whose sequence is more closely related to those of the DNA ligases encoded by poxviruses than to replicative DNA ligases, such as human DNA ligase I. In vitro transcription and translation of the cDNA produced a catalytically active DNA ligase similar in size and substrate specificity to the purified bovine enzyme. The DNA ligase III gene was localized to human chromosome 17, which eliminated this gene as a candidate for the cancer-prone disease Bloom syndrome that is associated with DNA joining abnormalities. DNA ligase III is ubiquitously expressed at low levels, except in the testes, in which the steady-state levels of DNA ligase III mRNA are at least 10-fold higher than those detected in other tissues and cells. Since DNA ligase I mRNA is also present at high levels in the testes, we examined the expression of the DNA ligase genes during spermatogenesis. DNA ligase I mRNA expression correlated with the contribution of proliferating spermatogonia cells to the testes, in agreement with the previously defined role of this enzyme in DNA replication. In contrast, elevated levels of DNA ligase III mRNA were observed in primary spermatocytes undergoing recombination prior to the first meiotic division. Therefore, we suggest that DNA ligase III seals DNA strand breaks that arise during the process of meiotic recombination in germ cells and as a consequence of DNA damage in somatic cells.

Coordinated activation of corneal wound response genes in vivo as observed by in situ hybridization.

We used subtractive screening of a cDNA library prepared from corneoscleral rims after cauterizing rat corneas. We identified 76 clones whose corresponding mRNA increased during the wound healing process in an in vivo model of injury which damages the corneal epithelium, stroma, and endothelium. Of these clones, 31 sequences encode known proteins. Another 45 clones are novel sequences based on comparison with the GenBank/EMBL databases. Changes in the level of expression of the novel genes, and a selected number of the known genes, were examined by in situ hybridization 22 and 72 hr after corneal injury. The majority produced a 'wound pattern' of expression such that the mRNAs were highly induced in all cell types adjacent to the wound site at 22 hr post injury. This signal decreased in intensity with distance from the wound site. In a subset of corneoslceral rims examined by in situ hybridization, the mRNAs for these genes were also highly induced in the limbal epithelium, where the progenitor corneal epithelial stem cells reside. By 72 hr, when acute tissue damage had been repaired, the induced mRNA was only faintly present in the thickened epithelium. Our results provide a useful framework for further studies defining the pathophysiological roles of the known and novel proteins encoded by the isolated cDNA clones.

Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.

DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.

Purification and characterization of DNA ligase III from bovine testes. Homology with DNA ligase II and vaccinia DNA ligase.

Mammalian cell nuclei contain three biochemically distinct DNA ligases. In the present study we have found high levels of DNA ligase I and DNA ligase III activity in bovine testes and have purified DNA ligase III to near homogeneity. The high level of DNA ligase III suggests a role for this enzyme in meiotic recombination. In assays measuring the fidelity of DNA joining, we detected no significant differences between DNA ligases II and III, whereas DNA ligase I was clearly a more faithful enzyme and was particularly sensitive to 3' mismatches. Amino acid sequences of peptides derived from DNA ligase III demonstrated that this enzyme, like DNA ligase II, is highly homologous with vaccinia DNA ligase. The absence of unambiguous differences between homologous peptides from DNA ligases II and III (10 pairs of peptides, 136 identical amino acids) indicates that these enzymes are either derived from a common precursor polypeptide or are encoded from the same gene by alternative splicing. Based on similarities in amino acid sequence and biochemical properties, we suggest that DNA ligases II and III, Drosophila DNA ligase II, and the DNA ligases encoded by the pox viruses constitute a distinct family of DNA ligases that perform specific roles in DNA repair and genetic recombination.

Rigid analogs of camptothecin as DNA topoisomerase I inhibitors.

Substituted 8-ethyl-2-(2-oxo-1,2-dihydroindol-3-ylidene)-8-hydroxy-2,3,5,8- tetrahydro-6-oxa-3a-azacyclopenta[b]naphthalene-1,4,7-triones were synthesized and evaluated as topoisomerase I inhibitors in an in vitro cleavable complex assay. The activity of these compounds may be attributed to their rigid, planar geometry, and an attempt was made to correlate the SAR in this series to known attributes of camptothecin.

Synthesis and antitumor activity of novel water soluble derivatives of camptothecin as specific inhibitors of topoisomerase I.

The synthesis and antitumor activities of the novel water soluble camptothecin derivatives 7-[(4-methylpiperazino)methyl]-10,11-(methylenedioxy)-(20S)-campto thecin trifluoroacetate (6) and 7-[(4-methylpiperazino)methyl]-10,11-(ethylenedioxy)-(20S)-camptot hecin trifluoroacetate (7) are described. The solubilities of compounds 6 and 7 were measured to be 4.5 and 5.8 mg/mL, respectively, in pH 5 acetate buffer in contrast to < 0.003 mg/mL for camptothecin in the same buffer. In the purified topoisomerase I cleavable complex enzyme assay, compounds 6 and 7 demonstrated potent inhibition of topoisomerase I with IC50's of 300 and 416 nM, respectively, in comparison to 679 nM for camptothecin and 1028 nM for topotecan. In human tumor cell cytotoxicity assays, compounds 6 and 7 demonstrated potent antitumor activity against ovarian (SKOV3), ovarian with upregulated MDRp-glycoprotein (SKVLB), melanoma (LOX), breast (T47D), and colon (HT29) with IC50's ranging from 0.5 to 102 nM. Compounds 6 and 7 induced tumor regressions in the HT29 human colon tumor xenograft model and demonstrated similar rank order of potency compared to in vitro assay results.

In vivo antitumor activity of two new seven-substituted water-soluble camptothecin analogues.

The development of camptothecin-like compounds as inhibitors of topoisomerase I for the treatment of resistant tumors has generated clinical excitement in this new class of drugs. We have developed two novel water-soluble camptothecin analogues which are specific inhibitors of topoisomerase I and are potent cytotoxins with significant antitumor activity. We added water-solubilizing groups off position 7 in the B ring of either 10,11-ethylenedioxy- or 10,11-methylenedioxy-20(S)-camptothecin. These water-soluble camptothecin analogues were demonstrated to be nanamolar inhibitors of the topoisomerase I enzyme in the cleavable complex assay. The compounds, GI147211 [7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)-camp tot hecin], and GI149893 [7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)-cam pto thecin], were compared to topotecan, a known water-soluble inhibitor of topoisomerase I. Both GI compounds were found to be slightly more potent than topotecan as inhibitors of topoisomerase I in the cleavable complex assay and were 1.5-2 times more soluble. Tumor cell cytotoxicity assays using 5 separate cell lines demonstrated that both GI compounds were 5-10 times more potent than topotecan, although by comparison all three topoisomerase I inhibitors were unaffected by the multidrug resistance P-glycoprotein. The antitumor activity of all three topoisomerase I inhibitors was compared concomitantly in two human colon xenograft models. In both models, GI147211 and GI149893 were able to induce regression of established HT-29 and SW-48 colon tumors by as much as 60%. The antitumor activity of both compounds were also demonstrated in the MX-1 and PC-3 xenografts. Microscopic examination of selected tissues indicated that drug-induced toxicity was primarily limited to the gastrointestinal tract and was comparable among the three compounds. Further clinical development of this class of compounds is ongoing.

Mammalian DNA ligase II is highly homologous with vaccinia DNA ligase. Identification of the DNA ligase II active site for enzyme-adenylate formation.

Mammalian cells contain three biochemically distinct DNA ligases. In this report we describe the purification of DNA ligase II to homogeneity from bovine liver nuclei. This enzyme interacts with ATP to form an enzyme-AMP complex, in which the AMP moiety is covalently linked to a lysine residue. An adenylylated peptide from DNA ligase II contains the sequence, Lys-Tyr-Asp-Gly-Glu-Arg, which is homologous to the active site motif conserved in ATP-dependent DNA ligases. The sequences adjacent to this motif in DNA ligase II are different from the comparable sequences in DNA ligase I, demonstrating that these enzymes are encoded by separate genes. The amino acid sequences of 15 DNA ligase II peptides exhibit striking homology (65% overall identity) with vaccinia DNA ligase. These peptides are also homologous (31% overall identity) with the catalytic domain of mammalian DNA ligase I, indicating that the genes encoding DNA ligases I and II probably evolved from a common ancestral gene. Since vaccinia DNA ligase is not required for DNA replication but influences the ability of the virus to survive DNA damage, the homology between this enzyme and DNA ligase II suggests that DNA ligase II may be involved in DNA repair.

Collagen cross-link metabolites in urine as markers of bone metastases in prostatic carcinoma.

The efficacy of radionuclide bone scans in monitoring metastatic bone activity remains controversial. Objective measurement of bone tumor burden would be useful for the evaluation of new therapies for metastatic carcinoma of the prostate. The recent discovery of the urinary excretion of pyridinoline (cross-link of mature collagen found in cartilage and bone) and deoxypyridinoline (collagen cross-link specific to bone) measured by high pressure liquid chromatography has provided sensitive specific indexes of cartilage and bone breakdown in rheumatoid arthritis, osteoporosis and metabolic bone diseases. We compared the urinary excretion of deoxypyridinoline,pyridinoline and hydroxyproline relative to urinary creatinine (nmol./mmol.creatinine) in 27 patients with benign prostatic hyperplasia (patient age 70.0 +/- 8.5 years, standard deviation), 29 with clinically confined prostate cancer (age 70.2 +/- 9.7 years), and 26 with prostate cancer and bone metastases (age 71.1 +/- 7.7 years). No diurnal variation of deoxypyridinoline or pyridinoline urinary excretion was detected in 5 patients with metastases. Urinary excretion of pyridinoline and deoxypyridinoline was significantly greater in patients with metastatic carcinoma of the prostate compared with patients with either benign prostatic hyperplasia (Mann-Whitney-Wilcoxon rank sum analysis, p < 0.00004 and 0.002, respectively) or localized prostate cancer (Mann-Whitney-Wilcoxon, p < 0.00001 and 0.00005, respectively). Urinary hydroxyproline levels failed to separate the 3 groups. Pyridinoline and deoxypyridinoline excretion in prostate cancer patients with metastases directly correlated with bone scan Soloway scores (r = 0.55, p < 0.005 and r = 0.57, p < 0.004 respectively), whereas serum prostate specific antigen did not (r = 0.36, p = 0.08). Serial measurements of pyridinoline and deoxypyridinoline progressively increased in 3 patients with clinical progression documented by new metastatic lesions by bone scan. Measurement of pyridinoline and deoxypyridinoline excretion cannot diagnose metastatic disease. However, these markers should be evaluated further for quantitative assessment of bone metastases.

Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: demonstration of tumor-type specificity and implications for cancer chemotherapy.

Topoisomerase I has been identified as an intracellular target of camptothecin, a plant alkaloid with anticancer activity. Various lines of evidence suggest that the sensitivity of cells to this drug is directly related to the topoisomerase I content. In humans, the levels of topoisomerase I have been shown to be elevated in colorectal tumors, compared to normal colon mucosa. The aim of our study was to determine whether (a) topoisomerase I levels are elevated in other solid tumors, (b) the elevated enzyme is catalytically active in these tumors, and (c) the increase in topoisomerase I levels in colorectal tumors is a result of increased transcription or translation. Topoisomerase I levels were quantitated in crude extracts from colorectal, prostate, and kidney tumors and their matched normal counterparts by Western blotting and by direct determination of catalytic activity, and mRNA levels were determined by Northern blotting. By Western blotting, colorectal tumors showed 5-35-fold increases in topoisomerase I levels, compared to their normal colon mucosa. In the case of prostate tumors, the increase was 2-10-fold, compared with benign hyperplastic prostate tissue from the same patients. However, no difference was observed in topoisomerase I levels in kidney tumors, compared to their normal counterparts. The catalytic activity of topoisomerase I was determined by a quantitative 32P-transfer assay in crude homogenates, without isolating nuclei. Colorectal and prostate tumors exhibited 11-40- and 4-26-fold increases, respectively, in catalytic activity. However, kidney tumors did not show any alteration in catalytic activity, compared to their normal matched samples. Thus, for all three tumor types there was a good correlation between enzyme levels and catalytic activity. Finally, colorectal tumors were analyzed for steady state mRNA levels. A 2-33-fold increase in mRNA levels was found in colorectal tumors, compared to normal colon mucosa. These results suggest that alterations in topoisomerase I expression in humans are tumor type specific and that the increase in topoisomerase I levels results from either increased transcription of the topoisomerase I gene or increased mRNA stability.

Plant antitumor agents. 30. Synthesis and structure activity of novel camptothecin analogs.

A large number of camptothecin (CPT) analogs have been prepared in the 20S, 20RS, and 20R configurations with a number of ring A substituents. Topoisomerase I (T-I) inhibition data (IC50) have been obtained by standard procedures. In general, substitution at the 9 or 10 positions with amino, halogeno, or hydroxyl groups in compounds with 20S configuration results in compounds with enhanced T-I inhibition. Compounds in the 20RS configuration were less active in vitro and in vivo and those in the 20R configuration were inactive. Compounds with 10,11-methylenedioxy substitution on ring A displayed a marked increase in potency in the T-I inhibition assay. The activities of some of the analogs as determined in a variety of in vivo assays including the L-1210 mouse leukemia assay were, in general, in accord with T-I inhibition. A number of water-soluble analogs such as 20-glycinate esters, 9-glycinamides, or hydrolyzed lactone salts were prepared and tested in in vitro and in vivo assays. In general, these compounds were less active than CPT both in terms of T-I inhibition and life prolongation in the L-1210 assay. However, certain 20-glycinate esters showed good in vivo activity after iv administration.