Designing a broad-spectrum integrative approach for cancer prevention and treatment.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.

eIF4E-Overexpression imparts perillyl alcohol and rapamycin-mediated regulation of telomerase reverse transcriptase.

Translation is mediated partly by regulation of free eukaryotic initiation factor 4E (eIF4E) levels through PI3K-Akt-mTOR signaling. Cancer cells treated with the plant-derived perillyl alcohol (POH) or the mechanistic target of rapamycin (mTOR) inhibitor rapamycin dephosphorylate eIF4E-binding protein (4E-BP1) and attenuate cap-dependent translation. We previously showed in cancer cell lines with elevated eIF4E that POH and rapamycin regulate telomerase activity through this pathway.

Disruption of an hTERT-mTOR-RAPTOR protein complex by a phytochemical perillyl alcohol and rapamycin.

We previously demonstrated in prostate cancer cells that a phytochemical-perillyl alcohol-and the mechanistic target of rapamycin (mTOR) inhibitor rapamycin rapidly attenuated telomerase activity. Protein levels of the telomerase catalytic subunit reverse transcriptase (hTERT) were diminished in the absence of an effect on hTERT mRNA, supporting an effect on 4E-BP1 phosphorylation and reduced initiation of protein translation. The decline in hTERT protein did not coincide wholly, however, with loss of telomerase activity suggesting a further level of regulation.

The isoprenoid perillyl alcohol inhibits telomerase activity in prostate cancer cells.

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell.

InTERTesting association between telomerase, mTOR and phytochemicals.

Telomeres are stretches of repeated DNA sequences located at the ends of chromosomes that are necessary to prevent loss of gene-coding DNA regions during replication. Telomerase - the enzyme responsible for immortalising cancer cells through the addition of telomeric repeats - is active in ~90% of human cancers. Telomerase activity is inhibited by various phytochemicals such as isoprenoids, genistein, curcumin, epigallocatechin-3-gallate, resveratrol and others.

Xeroderma pigmentosum variant: complementary molecular approaches to detect a 13 base pair deletion in the DNA polymerase eta gene.

Deficiencies of DNA polymerase eta-an enzyme mediating replication past UV-induced DNA damage-predispose individuals to xeroderma pigmentosum variant (XPV) and result in a high incidence of skin cancers. We designed, developed and assessed several complementary molecular approaches to detect a genetically inherited deletion within DNA polymerase eta. RNA was reverse transcribed from XPV fibroblasts and from normal human cells, and standard polymerase chain reaction (PCR) was conducted on the cDNA targeting a region with a 13 base pair deletion within the polymerase eta gene.

The cladribine conundrum: deciphering the drug's mechanism of action.

Importance Of The Field: Understanding fully the mechanism(s) of action of current and novel anticancer drugs is essential to optimize treatment regimens for oncology patients, to improve or extend drug efficacy and reduce patient side effects. Nucleoside analogues, either alone or in combination with additional therapeutic agents, are an essential part of first-line and salvage regimens directed against neoplastic diseases. However, many mechanistic studies on this class of drugs have been carried out in vitro or ex vivo at drug concentrations that are orders of magnitude higher than levels achieved in vivo.

Perillyl alcohol and genistein differentially regulate PKB/Akt and 4E-BP1 phosphorylation as well as eIF4E/eIF4G interactions in human tumor cells.

Previously we demonstrated that secondary products of plant mevalonate metabolism called isoprenoids attenuate 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA translational efficiency and cause tumor cell death. Here we compared effects of "pure" isoprenoids (perillyl alcohol and gamma-tocotrienol) and a "mixed" isoprenoid-genistein-on the PKB/Akt/mTOR pathway that controls mRNA translation and m(7)GpppX eIF4F cap binding complex formation. Effects were cell- and isoprenoid-specific.

Presence of the anti-leukemic nucleotide analog, 2-chloro-2'-deoxyadenosine-5'-monophosphate, in a promoter sequence alters DNA binding of TATA-binding protein (TBP).

2-Chlorodeoxyadenosine (CldAdo, Cladribine), a nucleoside analog used in the treatment of hairy cell leukemia, is phosphorylated and incorporated into DNA, but is not an absolute chain terminator. We hypothesized that the presence of a chlorine molecule projecting into the DNA minor groove would affect DNA:protein-binding interactions. Here, we investigated recognition of and binding to double-stranded CldAMP-substituted TATA promoter sequences by human TATA-binding protein (TBP) using mobility shift assays.

The antileukemia drug 2-chloro-2'-deoxyadenosine: an intrinsic transcriptional antagonist.

The nucleoside analog 2-chloro-2'-deoxyadenosine (CldAdo; cladribine) is effective in the treatment of hairy cell leukemia and chronic lymphocytic leukemia. CldAdo is phosphorylated and incorporated into cellular DNA but is not an absolute chain terminator. We demonstrated by in vitro gel-shift assays that binding interactions of the human TATA box-binding protein (TBP) were disrupted on 2-chlorodeoxyadenosine monophosphate (CldAMP)-substituted TATA box consensus sequences.

2-Chloro-2'-deoxyadenosine: alteration of DNA:TATA element binding protein (TBP) interactions.

2-Chloro-2'-deoxyadenosine (CldAdo, cladribine) is used therapeutically for hairy cell and other leukemias. The nucleoside is converted in leukemia cells to the 2-Cl-analog of dATP and incorporated into DNA, where it may alter binding of transcription factors to gene regulatory AT-rich sequences. Here we model the effects of CldATP incorporation into the adenovirus major late promoter TATA element recognized by TATA binding protein (TBP).

Nucleotide excision repair-deficient human cells in culture exhibit decreased survival after 2-chlorodeoxyadenosine treatment.

2-Chloro-2'-deoxyadenosine (CldAdo, cladribine) is a clinically important nucleoside analog for adult and pediatric leukemias. We previously described an activity in HeLa cell nuclear extracts that specifically recognized CldAMP-substituted oligomers. The factor was present in extracts prepared from repair-deficient xeroderma pigmentosum (XP) complementation group A cells, but not from group E--which are defective in damaged DNA-binding (DDB) protein--suggesting a possible repair process for incorporated analogs.

Sterol-independent regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in tumor cells.

Elevated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase expression supports synthesis of prenyl pyrophosphate intermediates required for tumor growth. In this study, the copy number of HMG-CoA reductase mRNA was determined in solid tumor and leukemic cell lines using competitive reverse transcriptase-polymerase chain reaction. Reductase mRNA was increased about eight-fold in Caco2 human colon adenocarcinoma cells compared with that in CCD18 normal colon cells.

Isoprenoid-mediated inhibition of mevalonate synthesis: potential application to cancer.

Pure and mixed isoprenoid end products of plant mevalonate metabolism trigger actions that suppress 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity. These actions modulate HMG CoA reductase mRNA translation and the proteolytic degradation of HMG CoA reductase. Such post-transcriptional events, we propose, are activated directly by acyclic isoprenoids and indirectly by cyclic isoprenoids.

Analysis of 2-chloro-2'-deoxyadenosine incorporation into cellular DNA by quantitative polymerase chain reaction.

Thermus aquaticus (Taq) DNA polymerase elongation is blocked by several DNA adducts. This property has been exploited in polymerase chain reaction (PCR) methods to analyze cellular DNA damage and repair after exposure to damaging agents. Such methods have not been applied previously to detect nucleoside analog incorporation into cellular DNA.

2-Chloro-2'-deoxyadenosine, an antileukemic drug, has an early effect on cellular mitochondrial function.

2-Chloro-2'-deoxyadenosine [CldAdo (cladribine)], a novel effective antileukemic agent, was examined for its effects on cellular mitochondrial function and DNA content after long term (< or = 7-day) incubation of cultured CCRF-CEM human leukemia cells. Dideoxycytidine (ddC), which is known to have a delayed effect on mitochondrial DNA content, was used as a positive control to monitor mitochondrial dysfunction. CldAdo at 6-16 nM was toxic to cells within 24 hr, which is in contrast to 300 nM ddC, which had no effect on cell growth for the first 4 days of treatment.

In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate.

2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction.

A human factor that recognizes DNA substituted with 2-chloroadenine, an antileukemic purine analog.

2-Chloro-2'-deoxyadenosine (cladribine), an analog of deoxyadenosine, is an important new drug for the treatment of hairy cell leukemia and other forms of adult and pediatric leukemia. By a gel-shift binding assay, we identified an activity in HeLa nuclear extracts that recognizes and binds to oligonucleotides substituted with 2-chloroadenine (ClAde). The activity was specific for ClAde residues because control oligomers did not readily compete out the complex.

2-Chloro-2'-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3'-->5' exonucleases.

2-Chloro-2'-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3'-->5' exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates.

Template 2-chloro-2'-deoxyadenosine monophosphate inhibits in vitro DNA synthesis.

2'-Chloro-2'-deoxyadenosine triphosphate (cladribine), a purine nucleotide analog and potent antileukemic agent, was enzymatically incorporated into 98-base oligomers in place of dATP to investigate the molecular consequences of 2-chloroadenine (CIAde) in DNA. We have used the resultant oligomers as templates for purified DNA polymerases, to compare the rate and extent of in vitro DNA synthesis; the sites of polymerase pausing, if any; and the effects of increasing deoxyribonucleoside triphosphate (dNTP) concentrations on synthetic reactions. Compared with control template, CIAde-containing DNA strikingly reduced the overall amount and rate of chain elongation by human polymerase beta and Klenow fragment.

Establishment of hypoxic conditions for cultured monolayer cells: hypoxia in five minutes or less.

A versatile deaeration chamber for altering the gaseous environment of monolayer cell cultures has been developed. The design is such that multiple samples of radioactively labeled or unlabeled cells can be gassed simultaneously with a short period of time. Deaeration times to reduce the level of oxygen in initially aerated cellular medium to very low levels (less than 20 parts per million) were decreased at least 6- to 20-fold when compared to most other methods commonly used to achieve N2- or N2O-induced hypoxia.

Polymerase chain reaction amplification of single-stranded DNA containing a base analog, 2-chloradenine.

By utilization of polymerase chain reaction techniques, single-stranded DNA of defined length and sequence containing a purine analog, 2-chloroadenine, in place of adenine was synthesized. This was accomplished by a combination of standard polymerase chain amplification reactions with Thermus aquaticus DNA polymerase in the presence of four normal deoxynucleoside triphosphates, M13 duplex DNA as template, and two primers to generate double-stranded DNA 118 bases in length. An asymmetric polymerase chain reaction, which produced an excess of single-stranded 98-base DNA, was then conducted with 2-chloro-2'-deoxy-adenosine 5'-triphosphate in place of dATP and with only one primer that annealed internal to the original two primers.

Effects of 2-chloroadenine substitution in DNA on restriction endonuclease cleavage reactions.

The purine analog, 2-chloro-2'-deoxyadenosine triphosphate (CldATP), was incorporated enzymatically in place of dATP into the minus strand of M13mp18 duplex DNA. Its effect on protein-DNA interactions was assessed by determining the amount of DNA cleavage by type II restriction endonucleases. Substitution of chloroadenine (CIAde) for adenine (Ade) in DNA appreciably decreased the amount and rate of DNA cleavage of the minus strand when the analog was situated within the appropriate endonuclease recognition site.

Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases.

2-Chloro-2'-deoxyadenosine 5'-triphosphate (CldATP) was compared with dATP as a substrate for DNA synthesis by bacterial and viral DNA polymerases in vitro. Lengths of chain extension and DNA synthesis pause sites were determined by comparison with products generated by dideoxynucleotide sequencing methods on the same end-labeled primer/template duplex after high-resolution polyacrylamide gel electrophoresis. Reverse transcriptase (RT) from human immunodeficiency virus (HIV-1) and avian myeloblastosis virus (AMV) incorporated CldATP efficiently.