Joseph G. Cory

5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to purple water-soluble formazans As cell-viability indicators

Abstract Analogs of MTT, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide, designed to yield water-soluble formazans upon reduction, have been synthesized and evaluated as cell-viability indicators.

Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): An inhibitor of ribonucleotide reductase with antineoplastic activity

The enzyme RR catalyzes the conversion of ribonucleoside diphosphates to their deoxyribonucleotide counterparts. RR is critical for the generation of the cytosine, adenine, and guanine deoxyribonucleotide 5-triphosphate building blocks of DNA, which are present in cells as exceedingly small intracellular pools. Therefore, interference with the function of RR might well result in an agent with significant antineoplastic activity, particularly against rapidly proliferating tumor cells. HUr is the only inhibitor of RR in clinical usage; this agent, however, is a relatively poor inhibitor of the enzyme and has a short serum half-life. Consequently, HUr is a relatively weak anticancer agent. In an effort to develop a more potent inhibitor of RR with utility as an anticancer agent, we have synthesized 3-AP and demonstrated (a) potent inhibition of L1210 leukemia cells in vitro, (b) curative capacity for mice bearing the L1210 leukemia, (c) marked inhibition of RR, and (d) sensitivity of HUr-resistant cells to 3-AP. These findings collectively demonstrate the clinical potential of 3-AP as an antineoplastic agent.

Inhibitors of ribonucleotide reductase. Comparative effects of amino- and hydroxy-substituted pyridine-2-carboxaldehyde thiosemicarbazones.

A new series of alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazones (HCTs) was studied for their effects on L1210 cell growth in culture, cell cycle transit, nucleic acid biosynthesis and ribonucleotide reductase activity. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) and 3-amino-4-methylpyridine-2-carboxaldehyde thiosemicarbazone (3-AMP) were the most active compounds tested with respect to inhibition of cell growth and ribonucleotide reductase activity. 5-Aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AP) and 4-methyl-5-aminopyridine-2-carboxaldehyde thiosemicarbazone (5-AMP) were slightly less active. 3-AP, 3-AMP, 5-AP and 5-AMP inhibited the incorporation of [3H]thymidine into DNA without affecting the rate of incorporation of [3H]uridine into RNA. The uptake and incorporation of [14C]cytidine into cellular ribonucleotides and RNA, respectively, were not decreased by 3-AP or 3-AMP; however, the incorporation of cytidine into DNA via ribonucleotide reductase was inhibited markedly. Thus, a pronounced decrease in the formation of [14C]deoxyribonucleotides from radioactive cytidine occurred in the acid-soluble fraction of 3-AP- and 3-AMP-treated L1210 cells. Consistent with an inhibition of DNA replication that occurred at relatively low concentrations of 3-AP and 3-AMP, cells gradually accumulated in the S-phase of the cell cycle; at higher concentrations of 3-AP and 3-AMP, a more rapid accumulation of cells in the G0/G1 phase of the cell cycle occurred, with the loss of the S-phase population, implying that a second less sensitive metabolic lesion was created by the HCTs. N-Acetylation of 3-AMP resulted in a compound that was 10-fold less active as an inhibitor of ribonucleotide reductase activity and 8-fold less active as an inhibitor of L1210 cell growth. N-Acetylation of either 5-AP or 5-AMP did not alter the inhibitory properties of these compounds. The results obtained provide an experimental rationale for the further development of the HCTs, particularly 3-AP and 3-AMP, as potential drugs for clinical use in the treatment of cancer.

Overexpression of the multidrug resistance genes mdr1, mdr3, and mrp in L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase

L1210 MQ-580 is a murine leukemia cell line resistant to the cytotoxic activity of the alpha-(N)-heterocyclic carboxaldehyde thiosemicarbazone class of inhibitors of ribonucleotide reductase. The line is cross-resistant to etoposide, daunomycin, and vinblastine. L1210 MQ-580 cells expressed 8-fold resistance to 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a relatively newly developed inhibitor of ribonucleotide reductase. The accumulation of [14C]3-AP by L1210 MQ-580 cells was 5- to 6-fold less than by parental L1210 cells. An increased rate of efflux of 3-AP was responsible for the lower steady-state concentration of 3-AP in resistant cells. In reverse transcription-polymerase chain reaction assays, L1210 MQ-580 cells were found to overexpress the multidrug resistance genes mdr1, mdr3, and mrp, but not the mdr2 gene, compared with parental L1210 cells. Measurement of the steady-state concentration of doxorubicin, a potential substrate for both the mdr and mrp gene products, demonstrated that L1210 MQ-580 cells accumulated 4-fold less anthracycline than parental cells. These findings indicate that drug efflux is a major determinant of the pattern of cross-resistance of L1210 MQ-580 cells. To extrapolate these observations to the human homologues of the mdr1, mdr3, and mrp murine genes, the effects of 3-AP were measured in L1210/VMDRC0.06 and NIH3T3 36-8-32 cells transfected with human MDR1 and MRP cDNAs, respectively. The transfectants were 2- to 3-fold resistant to the cytotoxic effects of 3-AP and accumulated less [14C]3-AP than their parental mock-transfected counterparts. Moreover, the cytotoxic activity of 3-AP was significantly greater in two double mrp gene knockout cell lines than in parental W 9.5 embryonic stem cells. Thus, the results suggest that 3-AP is a substrate for both the P-glycoprotein and MRP and that baseline MRP expression has the capacity to exert a protective role against the toxicity of this agent.

Structure-function relationships for a new series of pyridine-2-carboxaldehyde thiosemicarbazones on ribonucleotide reductase activity and tumor cell growth in culture and in vivo.

The synthesis of a new series of pyridine-2-carboxaldehyde thiosemicarbazones (HCTs) that have amino groups in the 3- and 5-positions has allowed the comparison of the structure/function relationships with regard to inhibition of ribonucleotide reductase activity, L1210 cell growth in culture and L1210 leukemia in vivo. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazones are more active than the corresponding 3-hydroxy-derivatives. The 3-amino-2-pyridine carboxaldehyde thiosemicarbazones were also more active then the 5-amino-2-carboxaldehyde thiosemicarbazones in inhibiting ribonucleotide reductase activity and L1210 cell growth in culture and in vivo. N-Acetylation of the 3-amino derivative resulted in a compound that was much less active both in vitro and in vivo; N-acetylation of the 5-amino derivative did not alter the in vitro inhibitory properties, but did eliminate the antitumor properties in vivo. When the most active HCTs were studied in more detail, it was found that the incorporation of [3H]thymidine into DNA was inhibited completely without the inhibition of [3H]uridine incorporation into RNA. Further, the conversion of [14C]cytidine to deoxycytidine nucleotides and incorporation into DNA was inhibited by the HCTs without an effect on the incorporation of cytidine into RNA. These data support the conclusion that ribonucleotide reductase is the major site of action of these HCTs. The 3-aminopyridine-2-carboxaldehyde thiosemicarbazones emerge as strong candidates for development for clinical trials in cancer patients.

2′-deoxy-2′-methylene derivatives of adenosine, guanosine, tubercidin, cytidine and uridine as inhibitors of L1210 cell growth in culture

The 2-deoxy-2-methylene derivatives of adenosine (MdAdo), guanosine (MdGuo), tubercidin (MdTu), cytidine (MdCyd) and uridine (MdUrd) were synthesized as mechanism-based inhibitors directed at ribonucleotide reductase. It was shown that MdCyd 5-diphosphate irreversibly inactivated ribonucleotide reductase from Escherichia coli (Baker et al., J Med Chem 34: 1879-1884, 1991). In studies reported here, MdAdo/EHNA, MdGuo and MdCyd inhibited L1210 cell growth with IC50 values of 3.4, 10.6 and 1.4 microM, respectively. Since MdAdo is a substrate for adenosine deaminase, the presence of EHNA was required to give maximal growth inhibition. 8-Aminoguanosine was not required to maximize the cytotoxic effects of MdGuo. The 2-deoxy-2-methylene derivatives of tubercidin and uridine did not inhibit L1210 cell growth at concentrations as high as 50 microM (MdTu) or 100 microM (MdUrd). L1210 cell lines resistant to hydroxyurea (directed at the non-heme iron subunit of ribonucleotide reductase) or deoxyadenosine (directed at the effector binding subunit of ribonucleotide reductase) were not resistant to MdCyd. An L1210 cell line that was highly resistant to dGuo due to the loss of a relatively specific deoxyribonucleoside kinase (Cory et al., J Biol Chem 268: 405-409, 1993) had a 6.6-fold increase in the IC50 value toward MdCyd, but showed only a 2-fold increase in resistance to MdGuo. Another L1210 cell line that was markedly deficient in adenosine kinase activity was highly resistant to MdAdo. Analysis by flow cytometry showed that MdCyd showed the transit of the cells through the G2/M phase of the cell cycle resulting in the buildup of the G2/M population. MdAdo, MdGuo and MdCyd inhibited the incorporation of [14C]cytidine into DNA without an effect on RNA synthesis or total cellular uptake of [14C]cytidine. The conversion of [14C]cytidine to deoxycytidine nucleotides was partially inhibited by MdGuo, but not by MdAdo or MdCyd. These data show that the 2-deoxy-2-methylene derivatives of adenosine, guanosine and cytidine are activated via specific nucleoside kinases and that the modes of action of these compounds are not identical.

Reduced level of ribonucleotide reductase R2 subunits increases dependence on homologous recombination repair of cisplatin-induced DNA damage.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) required for replicative and repair DNA synthesis. Mammalian RNR is a heteromeric enzyme consisting primarily of R1 and R2 subunits during the S phase of the cell cycle. We have shown previously that the presence of excess R2 subunits protects p53-deficient human colon cancer cells from cisplatin-induced DNA damage and replication stress. However, the mode of DNA repair influenced by changes in the level of the R2 subunit remained to be defined. In the present study, we demonstrated that depletion of BRCA1, an important factor of homologous recombination repair (HRR), preferentially sensitized stable R2-knockdown p53(−/−) HCT116 cells to the cytotoxicity of cisplatin and γ-H2AX induction. In accord with this finding, these R2-knockdown cells exhibited increased dependence on HRR, as evidenced by elevated levels of cisplatin-induced Rad51 foci and sister chromatid exchange frequency. Furthermore, stable knockdown of the R2 subunit also led to decreased cisplatin-induced gap-filling synthesis in nucleotide excision repair (NER) and a reduced dATP level in the G2/M phase of the cell cycle. These results suggest that an increased level of the R2 subunit extends the availability of dATP in the G2/M phase to promote the repair of NER-mediated single-strand gaps that are otherwise converted into double-strand breaks in the subsequent S phase. We propose that HRR becomes important for recovery from cisplatin-DNA lesions when the postexcision process of NER is restrained by reduced levels of the R2 subunit and dATP in p53-deficient cancer cells.

Lipophilic derivatives of cyclam as new inhibitors of tumor cell growth

Two new lipophilic tetraazamacrocycles were prepared and, in contrast to non-lipophilic analogs, found to be potent inhibitors of tumor cell growth in vitro with IC50 values below 10 micromolar.

Lack of competition of substrates for P-glycoprotein in MCF-7 breast cancer cells overexpressing MDR1

The MCF-7/Adr cells overexpress MDR-1 which contributes to the drug-resistant phenotype. Our studies show: 1. The retention of daunomycin in the MCF-7/Adr cells relates to a temperature-dependent and energy-dependent process. 2. The MCF-7/Adr cells retain less rhodamine-123 than the parental MCF-7 cells. 3. The MCF-7/Adr cells retain less daunomycin than the parental MCF-7 cells as measured by mean fluorescence or radioactive daunomycin. 4. Cyclosporin A and verapamil effectively block the effluxes of rhodamine-123 and daunomycin from the MCF-7/Adr cells. 5. On short-term incubation, 2-deoxyglucose lowers the NTP levels to a greater extent than sodium azide, showing the importance of glycolysis in the MCF-7 cell lines. 6. Although the MCF-7/Adr cells show cross-resistance to VP-16, actinomycin D and vinblastine, these drugs do not compete with daunomycin for the efflux pump. 7. These data suggest that either there must be multiple MDR-1 pumps that differ in substrate specificity or that there are distinct substrate sites on MDR-1.

Multifactorial mechanisms of drug resistance in tumor cell populations selected for resistance to specific chemotherapeutic agents

Abstract Mouse leukemia L1210 cells were treated with increasing concentrations of drugs that targeted the specific subunits of ribonucleotide reductase. From these studies mutant L1210 cell lines were selected that showed resistance to hydroxyurea (HU-R), deoxyadenosine (Y-8), MAIQ (MQ-580) and IMPY/deoxyadenosine (ED2). These cell lines had specific alterations that related directly to the ribonucleotide reductase site. These molecular changes included gene amplification, increased mRNA, increased enzyme activity and loss of sensitivity to dATP as a negative effector. However, each of the resistant cell lines did not have all of these changes. In addition to these changes, targeted at the ribonucleotide reductase site, the MQ-580, Y-8 and ED2 drug resistant cell lines had other alterations that are not readily attributable to the ribonucleotide reductase site. These changes included induced expression of MDR1, MRP (MQ-580), increased c-myc and c-fos expression (Y-8 and ED2), decreased p53 levels (Y-8 and ED2), increased frequency of CAD amplification (Y-8 and ED2), increased sensitivity to X-irradiation (Y-8) and a p53-independent pathway for apoptosis (Y-8). These data show that the development of drug resistance is complicated by the evolution of multifactorial mechanisms that may not be overtly related to the mechanism of action of the drug used to induce the resistance phenotype.