Combined proteasome and Bcl-2 inhibition stimulates apoptosis and inhibits growth in EBV-transformed lymphocytes: a potential therapeutic approach to EBV-associated lymphoproliferative diseases.

Objectives: Epstein-Barr virus (EBV) transforms B-cells into immortalized lymphoblastoid cells (LCLs) by triggering signaling pathways that lead to activation of multiple transcription factors and anti-apoptotic proteins, including NF-kappaB and Bcl-2, respectively. Since proteasome inhibition suppresses NF-kappaB activity, we sought to determine whether the proteasome inhibitor, bortezomib, alone or in combination with Bcl-2 inhibition, has potential as a therapeutic strategy in EBV-driven B-cell neoplasms.

Methods: We evaluated the effects of bortezomib in LCLs in vitro, in the presence and absence of the small molecular inhibitor of Bcl-2, HA14-1, on proliferation, apoptosis, caspase activation, and expression of Bcl-2 family members, and in vivo in the severe combined immunodeficiency (SCID) model of EBV+ lymphoproliferative disease.

Peptides inhibitory for the transcriptional regulatory function of human papillomavirus E2.

Purpose: Human papillomavirus (HPV) infections are associated with cervical neoplasia. Cellular and viral proteins are known to interact with the papillomavirus E2 protein to initiate transcription and DNA replication in the HPV life cycle. Our aim was to identify peptides that bind to the HPV16 E2 protein and thereby inhibit its ability to alter the transcriptional activity of other genes.

Coamplification of 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in methotrexate-resistant human leukemia cell lines.

Methotrexate (MTX)-resistant K562 human myelocytic leukemia sublines with 20- and 200-fold amplified dihydrofolate reductase (DHFR) genes localized to homogeneously staining regions (HSRs) on the long arms of chromosomes 5, 6, and 19 were used to examine whether other genes mapping close to the DHFR genes were coamplified. The gene for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, located on chromosome 5q13.3-14, was coamplified 4-14-fold, corresponding to the levels of resistance exhibited by these cells.

High and low levels of cottontail rabbit papillomavirus E2 protein generate opposite effects on gene expression.

The papillomavirus E2 protein plays an important role in viral transcriptional regulation and replication. We chose to study the cottontail rabbit papillomavirus (CRPV) E2 protein as a transcriptional regulator because of the availability of an animal model for papilloma formation, which may be relevant for human papillomavirus (HPV) infection and replication. We studied the effect of expression levels of E2 on the long control region, which contains transcriptional promoter and enhancer elements, and synthetic E2-dependent artificial promoters in which the E2 was the dominant factor in the transcriptional activation.

Increased rate of adenosine triphosphate-dependent etoposide (VP-16) efflux in a murine leukemia cell line overexpressing the multidrug resistance-associated protein (MRP) gene.

WEHI-3B/NOVO is a cloned murine leukemia cell line selected for resistance to novobiocin that is cross-resistant to the cytotoxic action of etoposide (VP-16) and to a lesser extent to a variety of other topoisomerase II (topo II)-reactive drugs. We have reported previously (Cancer Res. 52: 2782-2790, 1992) that WEHI-3B/NOVO cells exhibit a pronounced decrease in VP-16 induced DNA-topo II cross-link formation compared to the parental WEHI-3B/S cell line in intact cells, in the absence of a significant difference in the P4 unknotting activity of topo II assayed in nuclear extracts.

Sensitivity of K562 human chronic myelogenous leukemia blast cells transfected with a human multidrug resistance cDNA to cytotoxic drugs and differentiating agents.

The blast crisis of chronic myelogenous leukemia (CML) is refractory to most forms of cancer chemotherapy, but may be amenable to drugs that differentiate rather than kill leukemic cells. One mechanism implicated in resistance to cytodestructive drugs is overexpression of P-glycoprotein, the MDR1 gene product. While several classes of drugs sensitize multidrug-resistant (MDR) cells by interfering with the function of P-glycoprotein in vitro, few sensitizers have been effective in vivo.

Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis.

In the x-ray structure of the human dihydrofolate reductase, phenylalanine 31 and phenylalanine 34 have been shown to be involved in hydrophobic interactions with bound substrates and inhibitors. Using oligonucleotide-directed mutagenesis and a bacterial expression system producing the wild-type and mutant human dihydrofolate reductases at levels of 10% of the bacterial protein, we have constructed, expressed, and purified a serine 31 (S31) mutant and a serine 34 (S34) mutant. Fluorescence titration experiments indicated that S31 bound the substrate H2folate 10-fold tighter and the coenzyme NADPH 2-fold tighter than the wild-type human dihydrofolate reductase.

Bleomycin hydrolase: molecular cloning, sequencing, and biochemical studies reveal membership in the cysteine proteinase family.

Bleomycin (BLM) hydrolase catalyzes the inactivation of the antitumor drug BLM and is believed to protect normal and malignant cells from BLM toxicity. The normal physiological function of BLM hydrolase is not known. We now provide evidence for its membership in the cysteine proteinase family.

Amplification of a polymorphic dihydrofolate reductase gene expressing an enzyme with decreased binding to methotrexate in a human colon carcinoma cell line, HCT-8R4, resistant to this drug.

A methotrexate (MTX)-resistant human colon carcinoma cell line was obtained by growing HCT-8 cells in stepwise increasing concentrations of the drug. The resistant subline (HCT-8R4) was able to grow in the presence of 1 x 10(-4) M MTX and was found to have a 25-fold increase in the level of the target enzyme dihydrofolate reductase (DHFR), with a corresponding increase in DHFR gene copies as well as DHFR transcripts. Southern blot analysis of DNA from HCT-8R4 cells revealed the amplification of an altered gene.

Methotrexate resistant cells as targets for selective chemotherapy.

Strategies that are selective for eradicating methotrexate resistant cells are described. These strategies have been developed based on knowledge of the mechanism of drug resistance encountered in experimental systems and in the clinic. Drug resistance to methotrexate in experimental tumors is commonly due to either gene amplification (dihydrofolate reductase) or to impaired transport of methotrexate.

Molecular and karyological analysis of methotrexate-resistant and -sensitive human leukemic CCRF-CEM cells.

A methotrexate-resistant subline, CCRF-CEM/R1, was selected stepwise from the human leukemic lymphoblast T-cell line, CCRF-CEM, and maintained in 0.2 microM methotrexate. The development of resistance to methotrexate (75-fold) was associated with a 20-fold increase of dihydrofolate reductase activity.

Resistance to methotrexate due to gene amplification in a patient with acute leukemia.

A patient is described with acute myelocytic leukemia refractory to conventional therapy, who also became highly resistant to methotrexate (MTX) after repeated courses of this drug. Leukemia cells from this patient were found to contain an elevated level of dihydrofolate reductase (DHFR) activity, with no change in the affinity of the enzyme for MTX. A sensitive "dot blot" assay revealed a fourfold increase in the gene copy number of DHFR.

Amplification and organization of dihydrofolate reductase genes in a human leukemic cell line, K-562, resistant to methotrexate.

A subline of human leukemia cells (K-562), highly resistant to methotrexate, was developed by stepwise selection in the presence of increasing concentrations of this drug. The ED50 of these resistant cells was 1 mM compared to 10 nM for the parental line. Comparison of certain folate-requiring enzymes from crude extracts of the parent and resistant cells showed a 240-fold elevation of dihydrofolate reductase activity in the resistant cells with no significant increase in the levels of the other enzymes.

Gene amplification and altered enzymes as mechanisms for the development of drug resistance.

Two known mechanisms by which neoplastic cells may become resistant to chemotherapeutic agents are reviewed, using methotrexate (MTX) resistance as a model. These mechanisms are an increased level of target enzyme, found in several instances to be a consequence of gene amplification, or an altered target enzyme or receptor, less capable of binding the drug. An example of MTX resistance due to low-level gene amplification in leukemia cells from an MTX-resistant patient is described.