Expression microarray hybridization kinetics depend on length of the immobilized DNA but are independent of immobilization substrate.

Expression microarrays are often constructed by the immobilization of PCR products on two-dimensional modified glass slides or on three-dimensional microporous substrates. In this study we investigate whether the length of the immobilized species and the substrate choice influence hybridization dynamics. Using a simple bimolecular mass action controlled model to describe hybridization, we observed that the extent of hybridization and the initial velocities were directly dependent on the length of the immobilized species. An inflection point was noted at a length of 712 bases, above which the influence of length on hybridization rate decreased. Interestingly, we observed no differences in these parameters whether hybridization occurred on a two- or three-dimensional surface. Furthermore, the affinity of the solution phase labeled species for the immobilized species was identical for all arrayed lengths on both surfaces. These data indicate a similar interaction of the noncovalently immobilized species with either surface. Finally, we have determined that competitive hybridization on expression microarrays is nonlinear with respect to time and concentration of competitor. This observation is critical for analysis of expression array data.

FAST slides: a novel surface for microarrays.

We have evaluated FAST slides, a glass slide with a microporous polymeric surface that is a suitable substrate for microarray technology. The surface is a nitrocellulose-based polymer that binds DNA and proteins in a noncovalent but irreversible manner. FAST slides are compatible with robotic systems currently used to create microarrays and can easily accommodate volumes of 0.03-2 nL/spot. Our data indicate that FAST slides have a much higher binding capacity for DNA and better spot-to-spot consistency than traditional poly-lysine-coated slides. In addition, FAST slides are well suited for fluorescent detection because of their relatively low light scatter and efficient retention of arrayed DNA. These properties translate into fluorescent sensitivity comparable to modified glass surfaces. FAST slides are also ideal for arraying proteins, making them the only substrate of their kind currently available for microarray applications.

Cellular pharmacology of MTA: a correlation of MTA-induced cellular toxicity and in vitro enzyme inhibition with its effect on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

The mechanism of action of an antifolate may be investigated using a variety of experimental methods. These include experiments in a cell culture setting to observe possible protection against drug effects afforded by the end products of metabolic pathways, assessing the activity of purified target enzymes in the presence of the antifolate, and, finally, the measurement of drug effects on intracellular folate and nucleoside triphosphate pools. The current discussion is focused on studies using CCRF-CEM leukemia cells that were designed to compare and contrast mechanisms of action of the antifolates methotrexate, which is primarily a dihydrofolate reductase inhibitor, raltitrexed, a thymidylate synthase inhibitor, LY309887, a glycinamide ribonucleotide formyltransferase inhibitor, and MTA (multitargeted antifolate), which is a novel antifolate antimetabolite. The results of these studies support the hypothesis that MTA affects multiple enzymatic targets and has a distinct mechanism of action from methotrexate, raltitrexed, and LY309887.

Cell cycle modulation by a multitargeted antifolate, LY231514, increases the cytotoxicity and antitumor activity of gemcitabine in HT29 colon carcinoma.

The proliferation rate of HT29 colon carcinoma cells was decreased by the multitargeted antifolate (MTA), LY231514. This effect correlated with a buildup of cells near the G1-S interface after 24 h of incubation, and a synchronized progression of the population through S phase during the next 24 h. MTA treatment (0.03-3 microM) was minimally cytotoxic (20-30%) to HT29 cells after a 24-h exposure, and no dose response was observed. In contrast, the nucleoside analogue gemcitabine (GEM) was cytotoxic (IC50, 0.071 +/- 0.011 microM; IC90, 0.648 +/- 0.229 microM) after a 24-h exposure. We hypothesized that pretreatment of these cells with MTA would increase the potency of GEM by synchronizing the population for DNA synthesis. The cytotoxicity of GEM increased 2-7-fold when MTA was administered 24 h before GEM (IC50, 0.032 +/- 0.009 microM; IC90, 0.094 +/- 0.019 microM). In addition, an increase in cell kill for the combination compared with GEM alone (IC99, 12 microM for GEM alone; IC99, 0.331 microM for combination) was observed. No increase in potency or cell kill was observed when the two compounds were added simultaneously. MTA pretreatment also potentiated the cytotoxicity of a 1-h exposure to GEM. These cell-based observations were extended to evaluate the schedule-dependent interaction of these two agents in vivo using a nude mouse HT29 xenograft tumor model. At the doses tested, MTA alone (100 mg/kg) had a marginal effect on tumor growth delay, whereas GEM (80 mg/kg) produced a statistically significant tumor growth delay. In combination, the increase in tumor growth delay was greatest when MTA was administered before GEM, compared with simultaneous drug administration or the reverse sequence, e.g., GEM followed by MTA. The effect of sequential administration of MTA followed by GEM was greater than additive, indicating synergistic interaction of these agents. Thus, in vitro, MTA induced cell cycle effects on HT29 cells that resulted in potentiation of the cytotoxicity of GEM. In vivo, combination of these two drugs also demonstrated a schedule-dependent synergy that was optimal when MTA treatment preceded GEM.

The antiproliferative and cell cycle effects of 5,6,7, 8-tetrahydro-N5,N10-carbonylfolic acid, an inhibitor of methylenetetrahydrofolate dehydrogenase, are potentiated by hypoxanthine.

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.

Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.

A BRCA1 mutant alters G2-M cell cycle control in human mammary epithelial cells.

Mutations in BRCA1 increase the risk of breast and ovarian cancer. Although the mechanism by which mutant BRCA1 alters growth regulation is unknown, the COOH terminus of BRCA1 appears to play a critical role. To examine this, we introduced a vector expressing BRCA1 COOH-terminal residues 1293-1863 (CT-BRCA1) into nontumorigenic human breast epithelial cells. Overexpression of CT-BRCA1 led to a reduction in the doubling time (from 64 to 44 h) and a decreased reliance on growth factors, suggesting that this CT-BRCA1 may function in a dominant-negative manner. Expression of CT-BRCA1 induced alterations in cell cycle control, mainly in G2-M, including a loss of G2-M block by colchicine. These results suggest that one function of BRCA1-related growth control occurs by governing checkpoint(s) between DNA replication and mitosis.

Induction of phosphorylation on BRCA1 during the cell cycle and after DNA damage.

BRCA1, the familial breast cancer susceptibility gene product, is a 220-kDA phosphorylated protein. BRCA1 immunoprecipitated from MCF7 cells blocked in G1-S phase or progressing through S-phase of the cell cycle migrated more slowly through SDS polyacrylamide gels than BRCA1 from cells maintained in serum-supplemented media, serum-free media for 24 h, or delayed in G2-M phase by treatment with colchicine. Restoration of BRCA1 to the faster-migrating form, which occurred on release of cells from the G1-S-phase block, was prevented by the phosphatase inhibitor okadaic acid. Phosphatase treatment of immunoprecipitated BRCA1 resulted in the conversion of the slower-migrating form to the faster-migrating form. Although these results suggested that BRCA1 was preferentially hyperphosphorylated near the G1-S-phase boundary of the cell cycle, exposure of cells to DNA-damaging agents including UV light or treatment with hydrogen peroxide (H2O2) also promoted BRCA1 hyperphosphorylation. These same stimuli also eliminated the punctate nuclear staining pattern normally observed for BRCA1 in control cells. These results indicate that BRCA1 undergoes cyclic hyperphosphorylation during the cell cycle; however, this modification, as well as changes in BRCA1 nuclear staining, also occurs in response to DNA damage.

Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis.

PURPOSE: Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells were examined subsequent to inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) and prior to cell death or stasis. METHODS: Cell populations were treated with the GARFT inhibitors 6R-5, 10-dideazatetrahydrofolate (Lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments. RESULTS: Cell populations treated for up to 96h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G1, S and G2/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514, induced synchronization of the treatment population at the G1/S interface within 12h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24-48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells. CONCLUSION: These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.

Binding of phosphorothioate oligodeoxynucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent.

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant.

Cell surface binding and cellular internalization properties of suramin, a novel antineoplastic agent.

Although suramin has shown promise in preliminary clinical trials as an antineoplastic agent, it is unclear if its mode of action is predominately extracellular or intracellular. We have attempted to address this problem by studying the cellular pharmacology of tritiated suramin ([3H]suramin) in the DU145 and LNCaP prostate cancer cell lines, as well as in HL60 cells, an acute promyelocytic leukemia cell line. In the cell lines studied, significant, multisite, trypsin-insensitive, low-affinity cell surface binding by [3H]suramin was observed (Bmax > 10(6), Kd > 1 microM). The binding of [3H]suramin to the cell surface was competitive with respect to a phosphorothioate oligodeoxynucleotide homopolymer of cytidine, 28 bases in length, but was not affected by ATP. Use of this competitor allowed us to determine that [3H]suramin bound to the surface of HL60 cells was internalized via the process of adsorptive endocytosis and was maximal at approximately 6 h. In contrast, binding of suramin to the surface of the prostate cells, but not to that of HL60 cells, was completely abrogated by the presence of albumin (DU145 and LNCaP cells), or by warming to 37 degreesC (DU145 cells only). The dynamics of internalization and compartmentalization of suramin in DU145 revealed that within a narrow concentration range, internalization was dependent on time of exposure and drug concentration. Analysis of the exocytosis of suramin from DU145 cells revealed that approximately 64% of the drug was effluxed from a shallow compartment (t1/2 = 3.15 min) and 31% from a deep compartment (t1/2 = 433 min); both compartments probably represent endosomes. The results suggest that, because of the complexities of suramin's cellular pharmacology, its mechanism of action may vary signficantly according to cell type.

Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix.

We studied the interactions of phosphorothioate oligodeoxynucleotides and heparin-binding growth factors. By means of a gel mobility shift assay, we demonstrated that phosphodiester and phosphorothioate homopolymers bound to basic fibroblast growth factor (bFGF). Binding of a probe phosphodiester oligodeoxynucleotide could also be shown for other proteins of the FGF family, including acidic fibroblast growth factor (aFGF), Kaposi's growth factor (FGF-4) as well as for the bFGF-related vascular endothelial growth factor, VEGF. No binding to epidermal growth factor (EGF) was observed. In addition, using a radioreceptor assay, we have shown that phosphorothioate homopolymers of cytidine and thymidine blocked binding of not only 125I-bFGF, but also of 125I-PDGF to NIH 3T3 cells, whereas phosphodiester oligodeoxynucleotides were ineffective. The extent of blockade of binding was dependent on the chain length of the phosphorothioate oligodeoxynucleotide. Furthermore, we have examined the effects of 18-mer phosphorothioate oligodeoxynucleotides of different sequences on 125I-bFGF binding to low and high affinity sites on both NIH 3T3 fibroblasts and DU-145 prostate cancer cells. Despite the fact that we have observed inhibition of bFGF binding by the 18-mer phosphorothioate oligodeoxynucleotides for both the high and low affinity classes of bFGF receptor, the inhibition was sequence-selective only for the high affinity receptors. We have also demonstrated that phosphorothioate homopolymers of cytidine and thymidine release bFGF bound to low affinity receptors in extracellular matrix (ECM). Finally, the most potent phosphorothioate oligodeoxynucleotides used in these experiments (e.g. SdC28) were inhibitors of bFGF-induced DNA synthesis in NIH 3T3 cells.

Patterns of intracellular compartmentalization, trafficking and acidification of 5'-fluorescein labeled phosphodiester and phosphorothioate oligodeoxynucleotides in HL60 cells.

We have examined the intracellular compartmentalization and trafficking of fluorescein labeled (F) phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos) in HL60 cells. A series of F-oligos (PO and PS) were incubated for 6 hrs. with HL60 cells and the mean intracellular fluorescence determined by flow cytometry. The F signal was normalized by the addition of the ionophore monensin. An increase in signal intensity following addition of monensin indicated that the oligo was resident in an acidic intracellular environment. F-PS, but not F-PO oligos were found to reside in an acidic environment. An exception was a PO homopolymer of 15 cytidine bases (FOdC15) which was acidified. Using two different methods, the average resident intracellular pH of F-PS oligos and F-OdC15 was shown to be approximately 1 pH unit lower than that of F-PO oligos. Acidification of F-PS oligos could be blocked by the antibiotic bafilomycin, indicating that acidification was occurring in endosomes or vacuoles. F-PO and F-PS oligos were effluxed from HL60 cells from two intracellular compartments. However, approximately 60% of internalized F-PO oligo resided in a 'shallow' compartment that was turned over rapidly (t1/2 = 5-10 min.) whereas only 20% of F-PS oligo resided in this compartment. Conversely, approximately 80% of the internalized F-PS oligo but only 40% of F-PO oligo resided in a 'deep' compartment that turned over with t1/2 = 2-5 hrs. This report is the first quantitative demonstration that PO and PS oligos, and PO oligos of different sequences are trafficked differently by HL60 cells.

Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: a comparative study.

Phosphorodithioate (PS2) oligodeoxynucleotides (oligos) represent a relatively new class of backbone-modified oligo that have potential use as antisense agents. PS2 oligos are isoelectronic with phosphodiester (PO) and phosphoromonothioate (PS) oligos, and are nuclease resistant. However, unlike their PS congeners, PS2 oligos do not contain chiral centers. Little is known about the manner in which PS2 oligos interact with biological systems. In this study, we compare the cellular pharmacology of PS and PS2 oligos in HL60 cells. Cell surface binding, internalization, and compartmentalization are examined. Furthermore, the ability of PS and PS2 oligos to bind to rsCD4 and bFGF and to inhibit the activity of protein kinase C (PKC) is examined. Although the behavior of PS2 oligos closely parallels that of PS oligos, PS2 oligos appear to interact with some biological systems in a slightly different manner than PS oligos. These results indicate that PS2 oligos may have therapeutic potential other than as antisense agents.

Dynamics of the internalization of phosphodiester oligodeoxynucleotides in HL60 cells.

We have examined the cellular association and internalization of phosphodiester (PO) oligodeoxynucleotides (oligos) with HL60 cells. At 4 degrees C, a 15-mer PO homopolymer of thymidine (FOdT15) exhibits apparent saturation binding (Km = 22 +/- 1 nM) that is competitive with the binding of phosphorothioate (PS) oligos. The value of Kc for SdC28, a PS 28-mer homopolymer of cytidine, is 5 +/- 2 nM. SdC28 was used to strip cell surface fluorescence: Internalized fluorescence accumulated in a (concentration)(time)-dependent fashion, consistent with a pinocytotic mechanism. PS, and to a lesser extent, PO oligos inhibited the rate of internalization of fluorescent albumin, also a marker of pinocytosis. This was correlated with direct in vitro inhibition of protein kinase C (PKC) beta 1 by the PS and PO oligos. Furthermore, other PKC inhibitors (H7, staurosporine, DMSO, PKC pseudosubstrate polypeptide) also inhibited intracellular accumulation of pinocytosed materials, perhaps by stimulating the exocytosis rate. In HL60 cells, the pinocytotic internalization of charged oligos appears to be dependent on intact PKC kinase activity, which is inhibited in vitro by PS and PO oligos.

Stimulation of calcium influx in HL60 cells by cholesteryl-modified homopolymer oligodeoxynucleotides.

Cholesteryl-modified 15-mer homopolymers of cytidine and thymidine phosphodiester oligodeoxynucleotides (chol-OdC15 and chol-OdT15), but not chol-modified heteropolymeric oligos or chol-modified phosphorothioate oligos, were found to increase cytosolic free Ca2+ in HL60 cells. A flow cytometer and the calcium-sensitive dye indo-1 were used to make multiparameter measurements on the HL60 cells. Chol-OdC15 (5-10 microM) triggered a rapid increase (within 1 min) in [Ca2+]i, with a subsequent slow decline to baseline over 15 min in the continuous presence of agonist. The effect was preserved after unloading the intracellular Ca2+ stores with caffeine and ryanodine. The effect was not sensitive to membrane depolarization by KCl (60 mM) or nimodipine, a dihydropyridine calcium channel antagonist. An increase in [Ca2+]i was absent in a Ca(2+)-free solution and was inhibited by the inorganic Ca2+ channel blocker Cd2+. The results suggest that Ca2+ influx activated by the chol-oligomer is probably mediated by receptor-operated Ca2+ channels. This effect may be due to direct binding of the chol-oligo to the channel or to induced conformational changes due to modification of the local microenvironment.

Phosphorothioate oligodeoxynucleotides--anti-sense inhibitors of gene expression?

Phosphorothioate (PS) oligodeoxynucleotides are relatively nuclease resistant, water soluble analogs of phosphodiester (PO) oligodeoxynucleotides. These molecules are chiral but still hybridize well to their RNA targets. While considered for use as in vivo anti-sense inhibitors of gene expression, their biology, especially in the anti-viral area, is dominated by non-sequence specific effects. This review discusses both the sequence and non-sequence specific biologic effects of PS oligomers, and attempts to more clearly indicate their ultimate therapeutic potential.