Impact of genetic variability and treatment-related factors on outcome in early breast cancer patients receiving (neo-) adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide, and docetaxel.

To assess the impact of patient-related factors, including genetic variability in genes involved in the metabolism of chemotherapeutic agents, on breast cancer-specific survival (BCSS) and recurrence-free interval (RFI). We selected early breast cancer patients treated between 2000 and 2010 with 4-6 cycles of (neo-)adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide (FEC) or 3 cycles FEC followed by 3 cycles docetaxel. Tumor stage/subtype; febrile neutropenia and patient-related factors such as selected single nucleotide polymorphisms and baseline laboratory parameters were evaluated. Multivariable Cox regression was performed. Of 991 patients with a mean follow-up of 5.2 years, 152 (15.3 %) patients relapsed and 63 (6.4 %) patients died. Advanced stage and more aggressive subtype were associated with poorer BCSS and RFI in multivariable analysis (p < 0.0001). Associations with worse BCSS in multivariable analysis were: homozygous carriers of the rs1057910 variant C-allele in CYP2C9 (hazard ratio [HR] 30.4; 95 % confidence interval [CI] 6.1-151.5; p < 0.001) and higher white blood cell count (WBC) (HR 1.2; 95 % CI 1.0-1.3; p = 0.014). The GT genotype of the ABCB1 variant rs2032582 was associated with better BCSS (HR 0.5; 95 % CI 0.3-0.9, p = 0.021). Following associations with worse RFI were observed: higher WBC (HR 1.1; 95 % CI 1.0-1.2; p = 0.026), homozygous carriers of the rs1057910 variant C-allele in CYP2C9 (HR 10.9; 95 % CI 2.5-47.9; p = 0.002), CT genotype of the CYBA variant rs4673 (HR 1.8; 95 % CI 1.2-2.7; p = 0.006), and G-allele homozygosity for the UGT2B7 variant rs3924194 (HR 3.4; 95 % CI 1.2-9.7, p = 0.023). Patient-related factors including genetic variability and baseline white blood cell count, impacted on outcome in early breast cancer.

Genetic variability in the multidrug resistance associated protein-1 (ABCC1/MRP1) predicts hematological toxicity in breast cancer patients receiving (neo-)adjuvant chemotherapy with 5-fluorouracil, epirubicin and cyclophosphamide (FEC).

BACKGROUND: To assess the impact of single-nucleotide polymorphisms (SNPs) on predefined severe adverse events in breast cancer (BC) patients receiving (neo-)adjuvant 5-fluorouracil (FU), epirubicin and cyclophosphamide (FEC) chemotherapy. PATIENTS AND METHODS: Twenty-six SNPs in 16 genes of interest, including the drug transporter gene ABCC1/MRP1, were selected based on a literature survey. An additional 33 SNPs were selected in these genes, as well as in 12 other genes known to be involved in the metabolism of the studied chemotherapeutics. One thousand and twelve female patients treated between 2000 and 2010 with 3-6 cycles of (neo-)adjuvant FEC were genotyped for these SNPs using Sequenom MassARRAY. Severe adverse events were evaluated through an electronic chart review for febrile neutropenia (FN, primary end point), FN first cycle, prolonged grade 4 or deep (<100/µl) neutropenia, anemia grade 3-4, thrombocytopenia grade 3-4 and non-hematological grade 3-4 events (secondary end points). RESULTS: Carriers of the rs4148350 variant T-allele in ABCC1/MRP1 were associated with FN relative to homozygous carriers of the G-allele [P = 0.0006; false discovery rate (FDR) = 0.026]. Strong correlations with secondary end points such as prolonged grade 4 neutropenia (P = 0.002, FDR = 0.046) were also observed. Additionally, two other SNPs in ABCC1/MRP1 (rs45511401 and rs246221) correlated with FN (P = 0.007 and P = 0.01, respectively; FDR = 0.16 and 0.19), as well as two SNPs in UGT2B7 and FGFR4 (P = 0.024 and P = 0.04; FDR = 0.28 and 0.38). CONCLUSION: Genetic variability in ABCC1/MRP1 was associated with severe hematological toxicity of FEC.

CXCL12-CXCR4 axis in angiogenesis, metastasis and stem cell mobilization.

Chemokines are key players in the attraction and activation of leukocytes and are thus implicated in the recruitment of immune cells at sites of infection and/or inflammation. They exert their action by binding to seven-transmembrane G protein-coupled receptors. The chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 represents the single natural ligand for the chemokine receptor CXCR4. CXCL12 possesses angiogenic properties and is involved in the outgrowth and metastasis of CXCR4-expressing tumors and in certain inflammatory autoimmune disorders, such as rheumatoid arthritis. CXCR4 expression on tumor cells is upregulated by hypoxia and angiogenic factors, such as vascular endothelial growth factor (VEGF). CXCR4 also acts as a co-receptor for entry of human immunodeficiency virus (HIV) in CD4(+) T cells. Finally, CXCL12/CXCR4 interactions were shown to play an important role in the migration of hematopoietic stem cells and their progenitors from, and their retention within, the bone marrow, a site characterized by high CXCL12 expression. As such, CXCR4 inhibitors may be utilized to inhibit HIV-1 infection, tumor growth and metastasis and to mobilize hematopoietic stem cells from the bone marrow in the circulation, where they can be collected for autologous stem cell transplantation. Here, we discuss the different aspects of CXCL12/CXCR4 biology as well as the development and anti-cancer/stem cell mobilizing activity of CXCR4 antagonists.

X4 HIV-1 induces neuroblastoma cell death by interference with CXCL12/CXCR4 interaction.

Human neuroblastoma SK-N-SH cells strongly express CXC-chemokine receptor 4 (CXCR4), the principal coreceptor for X4 HIV-1 strains, and its natural ligand stromal cell-derived factor 1 (SDF-1, recently renamed CXCL12). We investigated the impact of CXCR4 blockade by the specific CXCR4 antagonist AMD3100 or by X4 HIV-1 virus particles on the growth and survival of neuroblastoma SK-N-SH cells. SK-N-SH cell proliferation was inhibited byAMD3100 and anti-CXCL12 neutralizing antibodies, but enhanced by exogenously added CXCL12. Upon prolongedexposure to AMD3100, SK-N-SH cell death occurred throughdeficit of survival-promoting and growth-stimulatory signals generated by endogenous CXCL12. In analogy with the observations made with the CXCR4 inhibitor AMD3100, the X4 HIV-1 strains IIIB and SF-2, but not the R5 strain BaL, caused a marked cytopathic effect and strongly effected SK-N-SH cell death after at least 10 days of incubation. However, no virus production could be detected in the HIV-1-inoculated SK-N-SH cell cultures. Exogenously added CXCL12 afforded partial protection against X4 HIV-1-induced cytopathicity in SK-N-SH cells. Our data indicate that the endogenous CXCL12/CXCR4 signaling axis is critical for neuroblastoma cell survival and proliferation. Long-term blockade of CXCR4 through physical contact with the X4 HIV-1 envelope can cause neuronal cell death. This mechanism may possibly play a role in X4 HIV-associated neurodegeneration.

Role of MRP4 and MRP5 in biology and chemotherapy.

Nucleotide efflux (especially cyclic nucleotides) from a variety of mammalian tissues, bacteria, and lower eukaryotes has been studied for several decades. However, the molecular identity of these nucleotide efflux transporters remained elusive, despite extensive knowledge of their kinetic properties and inhibitor profiles. Identification of the subfamily of adenosine triphosphate (ATP) binding cassette transporters, multidrug resistance protein (MRP) subfamily, permitted rapid advances because some recently identified MRP family members transport modified nucleotide analogs (ie, chemotherapeutic agents). We first identified, MRP4, based on its ability to efflux antiretroviral compounds, such as azidothymidine monophosphate (AZT-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA), in drug-resistant and also in transfected cell lines. MRP5, a close structural homologue of MRP4 also transported PMEA. MRP4 and MRP5 confer resistance to cytotoxic thiopurine nucleotides, and we demonstrate MRP4 expression varies among acute lymphoblastic leukemias, suggesting this as a factor in response to chemotherapy with these agents. The ability of MRP4 and MRP5 to transport 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) suggests they may play a biological role in cellular signaling by these nucleotides. Finally, we propose that MRP4 may also play a role in hepatic bile acid homeostasis because loss of the main bile acid efflux transporter, sister of P-glycoprotein (SPGP) aka bile-salt export pump (BSEP), leads to a strong compensatory upregulation in MRP4 expression. Cumulatively, these studies reveal that the ATP-binding cassette (ABC) transporters MRP4 and MRP5 have a unique role in biology and in chemotherapeutic response.

AMD3100, a potent and specific antagonist of the stromal cell-derived factor-1 chemokine receptor CXCR4, inhibits autoimmune joint inflammation in IFN-gamma receptor-deficient mice.

Autoimmune collagen-induced arthritis (CIA) in IFN-gammaR-deficient DBA/1 mice was shown to be reduced in severity by treatment with the bicyclam derivative AMD3100, a specific antagonist of the interaction between the chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4. The beneficial effect of the CXCR4 antagonist was demonstrable when treatment was initiated between the time of immunization and appearance of the first symptoms. Treatment also reduced the delayed-type hypersensitivity response to the autoantigen, collagen type II. These observations are indicative of an action on a late event in the pathogenesis, such as chemokine-mediated attraction of leukocytes toward joint tissues. The notion of SDF-1 involvement was further supported by the observation that exogenous SDF-1 injected in periarthritic tissue elicited an inflammatory response that could be inhibited by AMD3100. The majority of leukocytes harvested from inflamed joints of mice with CIA were found to be Mac-1(+) and CXCR4(+), and AMD3100 was demonstrated to interfere specifically with chemotaxis and Ca(2+) mobilization induced in vitro by SDF-1 on Mac-1(+)/CXCR4(+) splenocytes. We conclude that SDF-1 plays a central role in the pathogenesis of murine CIA, by attracting Mac-1(+)/CXCR4(+) cells to the inflamed joints.

Mutation of Asp(171) and Asp(262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100.

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investigated the interaction of AMD3100 with CXCR4 at the molecular level by mutational analysis. We established a set of stably transfected U87.CD4 cell lines expressing different mutant forms of CXCR4 (i.e., CXCR4[WT], CXCR4[D171N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the activity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4--as assessed by the inhibitory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5--was greatly reduced by substitution of Asp(171) and/or Asp(262) by neutral asparagine residue(s). Both aspartates, but most particularly Asp(262), also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His(281) by a neutral alanine potentiated the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D262N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domains 4 and 6 of the 7-transmembrane receptor, respectively, may represent crucial sites for electrostatic interaction of the positive charges of the bicyclams, as well as for the highly basic V3 loop of the gp120 envelope protein of certain HIV-1 strains.

Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs.

Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells. The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5. We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells. This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5. In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment. Inhibitors of organic anion transport inhibit transport mediated by MRP5. We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.

The multifunctional deoxynucleoside kinase of insect cells is a target for the development of new insecticides.

The antiherpetic agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was found to be an efficient substrate for recombinant Drosophila melanogaster-deoxyribonucleoside kinase with a K(m) of 4.5 microM and a V(max) of 400 nmol/microg protein/h compared with 1.3 microM and 62.5 nmol/microg protein/h, respectively, for the natural substrate thymidine. Mammalian cytosolic thymidine kinase-1 does not recognize BVDU as a substrate. In sharp contrast to mammalian cells, the insect D. melanogaster and Spodoptera frugiperda (Sf) embryonic cells proved highly sensitive to the cytostatic action of BVDU. BVDU was efficiently metabolized to its 5'-mono-, 5'-di- and 5'-triphosphate derivatives in the insect cell cultures and abundantly incorporated into the insect cell DNA. BVDU prevented the D. melanogaster cells to initiate the S phase of their cell cycle, and exposure of S. frugiperda cells to BVDU led to a dose-dependent retardation of the insect cells in the S phase of their cell cycle. Both inhibition of nucleic acid synthesis (through the 5'-triphosphate of BVDU) and inhibition of thymidylate synthase (through the 5'-monophosphate of BVDU) would account for the cytostatic activity of BVDU against the insect cells. Because of the virtual lack of cytotoxicity of BVDU against mammalian cells, the drug should be considered highly selective in its cytostatic action against the insect cells. When added to the food of S. frugiperda larvae, BVDU caused a remarkable decrease in the weight gain of the larvae and heavily compromised the transformation of the larvae to the pupae and their subsequent adult (moth) phase. Our data indicate that insect multifunctional deoxyribonucleoside kinase should be considered an entirely novel and attractive target in the development of new nucleoside types of highly selective insecticidal drugs.

CXC-chemokine receptor 4 is not a coreceptor for human herpesvirus 7 entry into CD4(+) T cells.

Human herpesvirus 7 (HHV-7) is a T-lymphotropic virus which utilizes the CD4 receptor as its main receptor to enter the target cells. Hence, HHV-7 can interfere with human immunodeficiency virus type 1 (HIV-1) infection in CD4(+) T cells. It was recently suggested that the CXC chemokine receptor 4 (CXCR4), which was found to be a crucial coreceptor for T-tropic HIV-1 strains, may also play a role in the HHV-7 infection process. However, the results presented here demonstrate that CXCR4 is not involved in HHV-7 infection. The natural ligand of CXCR4, SDF-1alpha, was not able to inhibit HHV-7 infection in SupT1 cells or in CD8(+) T-cell-depleted peripheral blood mononuclear cells. Also, AMD3100, a specific CXCR4 antagonist with potent antiviral activity against T-tropic HIV strains (50% inhibitory concentration ¿IC(50), 1 to 10 ng/ml), completely failed to inhibit HHV-7 infection (IC(50), >250 microg/ml). Thus, two different agents known to specifically interact with CXCR4 were not able to inhibit HHV-7 infection. Other T-lymphoid cell lines, expressing both CD4 and CXCR4 (e.g., HUT-78 and MT-4) could not be infected by HHV-7. In addition, the CD4-transfected cell lines HOS. CD4 and U87.CD4 and the CD4/CXCR4 double-transfected cell lines HOS. CD4.CXCR4 and U87.CD4.CXCR4 were not infectable with HHV-7. Also, we found no down-regulation of surface-bound or intracellular CXCR4 in HHV-7-infected CD4(+) T cells. As compared to uninfected SupT1 cells, stromal cell-derived factor 1alpha (SDF-1alpha)/CXCR4-mediated intracellular calcium flux was unchanged in SupT1 cells that were acutely or persistently infected with HHV-7. All these data argue against CXCR4 as a receptor involved in the HHV-7 infection process.

A second target for the peptoid Tat/transactivation response element inhibitor CGP64222: inhibition of human immunodeficiency virus replication by blocking CXC-chemokine receptor 4-mediated virus entry.

The peptoid CGP64222 has been previously demonstrated to inhibit the human immunodeficiency virus (HIV) Tat/transactivation response element complex formation. It has previously been shown that CGP64222 selectively inhibits HIV-1 long terminal repeat-driven gene expression and HIV-1(LAV) replication in lymphocytes. Here, we show that CGP64222 inhibits the replication of a wide range of laboratory strains of HIV-1 and HIV-2 in MT-4 cells. However, CGP64222 proved inactive in MT-4 cells against HIV-1 strains that are resistant to the bicyclams. The bicyclams are known to specifically interact with CXC-chemokine receptor 4, the main coreceptor used by T-tropic HIV strains to enter the cells. Mechanism of action studies revealed that CGP64222 can inhibit the HIV replicative cycle, also through a selective interaction with the CXC-chemokine receptor 4 coreceptor.

Mechanistic study on the cytostatic and tumor cell differentiation-inducing properties of 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir)-collected publications.

The therapeutic potential of the antiretroviral drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir) for the treatment of human immunodeficiency virus (HIV)- and human hepatitis B virus (HBV) infections is currently being explored in advanced clinical trials. In the present study, we investigated the impact of PMEA on cellular functioning (cell cycle, nucleotide metabolism, nucleic acid synthesis, ...). Moreover, we have unraveled the molecular/biochemical basis underlying the marked differentiation-inducing activity of PMEA (and related analogues) in tumor cells. We could demonstrate that PMEA is endowed with potent antitumor activity in a highly aggressive in vivo tumor model. These findings open new perspectives for the possible application of this type of compounds in cancer chemotherapy. The fact that AIDS patients frequently develop certain types of differentiation-susceptible malignancies (e.g. Kaposi's sarcoma) further attests the clinical relevance of our observations.

9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine: a novel prodrug of 9-(2-phosphonylmethoxyethyl)guanine with improved antitumor efficacy and selectivity in choriocarcinoma-bearing rats.

The novel acyclic nucleoside phosphonate analogue 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPr-PMEDAP) was shown in vitro to act as an intracellular prodrug of 9-(2-phosphonylmethoxyethyl)guanine (PMEG). We compared the in vivo antitumor efficacy and selectivity of cPr-PMEDAP, its progenitor PMEDAP, and PMEG in a rat choriocarcinoma tumor model. The rats, inoculated with rat choriocarcinoma (RCHO) cells under the renal capsule, were treated IP during 10 days. Macroscopical and histological examination of the RCHO-inoculated kidneys was performed at two time points (i.e., immediately after the end of treatment or after an additional drug-free period of 2 weeks). Complete inhibition of choriocarcinoma tumor development was achieved upon treatment with cPr-PMEDAP, PMEG, and PMEDAP at a daily dose of 10, 1, and 50 mg/kg, respectively. At these doses, all three compounds produced moderate to strong toxicity (evidenced by atrophy of lymphoid organs and reduced body weight gain). When compared at the maximum tolerated (sublethal) doses (i.e., 0.5, 10, and 50 mg/kg for PMEG, cPr-PMEDAP, and PMEDAP, respectively), cPr-PMEDAP proved superior to PMEG and PMEDAP in achieving a complete inhibition of tumor development. Also, whereas PMEG was unable to produce a prolonged antitumor effect, the animals treated with cPr-PMEDAP still showed prominent inhibition of tumor development when tumor size was evaluated at 2 weeks after end of treatment. Based on its efficacy and therapeutic safety, cPr-PMEDAP can be regarded as a promising antitumor agent, which merits further in vivo evaluation in additional tumor models for human neoplasms.

Antitumor potential of acyclic nucleoside phosphonates.

Acyclic nucleoside phosphonates such as HPMPC (cidofovir) and PMEA (adefovir) have been identified as broad-spectrum antiviral agents that are effective against herpes-, retro- and hepadnavirus infections (PMEA) and herpes-, pox-, adeno-, polyoma-, and papillomavirus infections (HPMPC). Here we show that HPMPC and PMEA also offer great potential as antitumor agents, through the induction of tumor cell differentiation (PMEA), inhibition of angiogenesis (HPMPC) and induction of apoptosis (HPMPC). In vivo tumor regressions have been noted for choriocarcinoma (PMEA) in rats, hemangioma (HPMPC) in rats and papillomatous lesions (HPMPC) in humans. Acyclic nucleoside phosphonates can be considered as a new dimension to the discipline of chemotherapy. They have a unique mode of action that is targeted at (viral or tumoral) DNA synthesis. They exhibit a pronounced and prolonged anti-viral and/or tumoral activity that can persist for days or weeks after a single administration. Most importantly, they have a uniquely broad spectrum of indications for clinical use, encompassing both DNA- and retrovirus infections, as well as various forms of cancer of both viral and non-viral origin.

Role of antimetabolites of purine and pyrimidine nucleotide metabolism in tumor cell differentiation.

Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP synthetase, thymidylate synthase, dihydrofolate reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase, and DNA methyltransferase, are markedly up-regulated in certain tumor cells. Together with the concomitant down-modulation of the purine and pyrimidine degradation enzymes, the increased anabolic propensity supports the excessive proliferation of transformed cells. However, many types of cancer cells have maintained the ability to differentiate terminally into mature, non-proliferating cells not only in response to physiological receptor ligands, such as retinoic acid, vitamin D metabolites, and cytokines, but also following exposure to a wide variety of non-physiological agents such as antimetabolites. Interestingly, induction of tumor cell differentiation is often associated with reversal of the transformation-related enzyme deregulations. An important class of differentiating compounds comprises the antimetabolites of purine and pyrimidine nucleotide metabolism and nucleic acid synthesis, the majority being structural analogs of natural nucleosides. The CTP synthetase inhibitors cyclopentenylcytosine and 3-deazauridine, the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine, the dihydrofolate reductase inhibitor methotrexate, the IMP dehydrogenase inhibitors tiazofurin, ribavirin, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and mycophenolic acid, the ribonucleotide reductase inhibitors hydroxyurea and deferoxamine, and the DNA polymerase inhibitors ara-C, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and aphidicolin, as well as several nucleoside analogs perturbing the DNA methylation pattern, have been found to induce tumor cell differentiation through impairment of DNA synthesis and/or function. Thus, by selectively targeting those anabolic enzymes that contribute to the neoplastic behavior of cancer cells, the normal cellular differentiation program may be reactivated and the malignant phenotype suppressed.

9-(2-Phosphonylmethoxyethyl)adenine induces tumor cell differentiation or cell death by blocking cell cycle progression through the S phase.

In addition to its inhibitory activity against viral DNA polymerases and reverse transcriptase, the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) also markedly inhibits the replicative cellular DNA polymerases alpha, delta, and epsilon. We have previously shown that PMEA is a strong inducer of differentiation in several in vitro tumor cell models and has marked antitumor potential in vivo. To elucidate the molecular mechanism of the differentiation-inducing activity of PMEA, we have now investigated the effects of the drug on cell proliferation and differentiation, cell cycle regulation, and oncogene expression in the human erythroleukemia K562 cell line. Terminal, irreversible erythroid differentiation of PMEA-treated K562 cells was evidenced by hemoglobin production, increased expression of glycophorin A on the K562 cell membrane, and induction of acetylcholinesterase activity. After exposure to PMEA, K562 cell cultures displayed a marked retardation of S-phase progression, leading to a severe perturbation of the normal cell cycle distribution pattern. Whereas no substantial changes in c-myc mRNA levels and p21, PCNA, cdc2, and CDK2 protein levels were noted in PMEA-treated K562 cells, there was a marked accumulation of cyclin A and, most strikingly, cyclins E and B1. A similar picture of cell cycle deregulation was also observed in PMEA-exposed human myeloid THP-1 cells. However, in contrast to the strong differentiation-inducing activity of PMEA in K562 cells, the drug completely failed to induce monocytic maturation of human myeloid THP-1 cells. On the contrary, THP-1 cells underwent apoptotic cell death in the presence of PMEA, as demonstrated by prelytic, intracellular DNA fragmentation and the binding of annexin V to the cell surface. We hypothesize that, depending on the nature of the tumor cell line, PMEA triggers a process of either differentiation or apoptosis by the uncoupling of normally integrated cell cycle processes through inhibition of DNA replication during the S phase.

N6-cyclopropyl-PMEDAP: a novel derivative of 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP) with distinct metabolic, antiproliferative, and differentiation-inducing properties.

N6-Cyclopropyl-PMEDAP (cPr-PMEDAP) is a novel derivative of the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (PMEDAP). Its cytostatic activity was found to be 8- to 20-fold more pronounced than that of PMEDAP and equivalent to that of the guanine derivative 9-(2-phosphonylmethoxyethyl)guanine (PMEG) against a variety of tumor cell lines. Unlike PMEDAP, but like PMEG, cPr-PMEDAP was equally cytostatic to wild-type and 9-(2-phosphonylmethoxyethyl)adenine/PMEDAP-resistant variants of the human erythroleukemia K562 and the murine leukemia L1210 cell lines. Also, cPr-PMEDAP and PMEG proved to be equipotent inducers of K562 and rat choriocarcinoma RCHO cell differentiation, whereas the differentiation-inducing activity of PMEDAP was 5- to 25-fold less pronounced. Furthermore, compared to PMEDAP, cPr-PMEDAP and PMEG were 10- to 25-fold more potent in inhibiting the progression of K562 cells through the S phase of the cell cycle, resulting in a marked accumulation of the four 2'-deoxyribonucleoside 5'-triphosphate pools. The biological effects of cPr-PMEDAP, but not PMEDAP, were reversed by the adenylate deaminase inhibitor 2'-deoxycoformycin (dCF). Formation of the deaminated derivative of cPr-PMEDAP (i.e. PMEG) was demonstrated in crude extracts from K562 and L1210 cells and in metabolism studies with radiolabeled cPr-PMEDAP and PMEG. This is the very first example of an acyclic nucleoside phosphonate analogue that is susceptible to deamination. However, cPr-PMEDAP was not recognized as a substrate by purified adenosine deaminase or by adenylate deaminase. These findings might point to an as yet unidentified cellular enzyme, sensitive to dCF but different from the common adenosine and AMP deaminases. Our data demonstrate the superior antiproliferative and differentiation-inducing effects of cPr-PMEDAP on tumor cells, as compared to the parent compound PMEDAP, based on the unique metabolic properties of this novel compound.

Impact of 9-(2-phosphonylmethoxyethyl)adenine on (deoxy)ribonucleotide metabolism and nucleic acid synthesis in tumor cells.

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.

Enhanced 9-(2-phosphonylmethoxyethyl)adenine secretion by a specific, indomethacin-sensitive efflux pump in a mutant 9-(2-phosphonylmethoxyethyl)adenine-resistant human erythroleukemia K562 cell line.

We have investigated the molecular basis of the 100-fold resistance of mutant human erythroleukemia K562/PMEA-1 cells to the antiproliferative potential of 9-(2-phosphonylmethoxyethyl)adenine (PMEA). Upon exposure to high PMEA concentrations, comparable intracellular PMEA levels were initially observed in mutant K562/PMEA-1 and wild-type K562/0 cells, indicating that PMEA influx was unaltered. However, after 4 hr of exposure to 0.2 microM [3H]bis(pivaloyloxymethyl)-PMEA [bis(POM)-PMEA], the total intracellular level of unphosphorylated and mono- and diphosphorylated PMEA was 2.8-fold lower in K562/PMEA-1 than in K562/0 cells. Increased PMEA secretion from K562/PMEA-1 cells (compared with K562/0 cells) became more pronounced upon prolonged exposure to bis(POM)-PMEA; after 24 hr, K562/PMEA-1 cells showed 65-fold lower total intracellular PMEA levels than K562/0 cells and at 48 hr, >400-fold less total PMEA was detected in K562/PMEA-1 cells. In addition, PMEA phosphorylation was 25- to 50-fold less efficient in K562/PMEA-1 than in K562/0 cells, pointing to an additional defect at the level of the metabolism of PMEA. The PMEA efflux mechanism was shown to be temperature- and azide-dependent, was markedly inhibited by indomethacin, and did not recognize adenine nucleotides or the phosphorylated metabolites of 3'-azido-3'-deoxythymidine. Also, over a 28-hr period, PMEA efflux was not affected by an inhibitor of RNA synthesis (actinomycin D) or protein synthesis (cycloheximide). Our studies revealed that resistance of K562/PMEA-1 cells to PMEA is the combined result of a severely impaired PMEA phosphorylation on the one hand, and an enhanced PMEA secretion by a highly specific, indomethacin-sensitive efflux pump, different from the classical P-glycoprotein- and multidrug resistance protein-mediated resistance mechanisms, on the other hand.

Potent differentiation-inducing properties of the antiretroviral agent 9-(2-phosphonylmethoxyethyl) adenine (PMEA) in the rat choriocarcinoma (RCHO) tumor cell model.

9-(2-phosphonylmethoxyethyl)adenine (PMEA) and its closely related structural analogue (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) are potent inhibitors of retroviruses and hepatitis B virus. In its oral prodrug form (adefovir dipivoxil), PMEA is currently the subject of advanced phase II/III clinical trials for the treatment of HIV infections. PMEA has also been shown to be a potent differentiation-inducing agent. In the present study, PMEA was found to have a strong differentiation-inducing effect on rat choriocarcinoma (RCHO) cells, comparable to that of methotrexate, which is the drug of choice for the chemotherapy of choriocarcinoma in humans. PMEA induced differentiation of choriocarcinoma trophoblast cells in a concentration-dependent manner within the 2- to 50-microM concentration range, as ascertained by giant cell formation, alkaline phosphatase induction, progesterone secretion, and the disappearance of a cytotrophoblast-specific surface antigen. PMEA had to be exposed to the rat choriocarcinoma cell cultures for at least 2-3 days to achieve optimal growth inhibition and differentiation of the tumor cells. Unlike PMEA, (R)-9-(2-phosphonylmethoxypropyl)adenine failed to induce differentiation of proliferating cytotrophoblasts into nonproliferating, hormonally active giant cells. This points to the specificity of PMEA as an inducer of choriocarcinoma cell differentiation.