Crizotinib induces apoptosis and gene expression changes in ALK+ anaplastic large cell lymphoma cell lines; brentuximab synergizes and doxorubicin antagonizes.

Background: Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) shows 60-70% event free survival with standard treatments. Targeted therapies are being tested for increased benefit and/or reduced toxicity, but interactions with standard agents are not well known.

Methods: We exposed four ALCL cell lines to two targeted agents, crizotinib and brentuximab vedotin, and to two standard agents, doxorubicin and vinblastine.

Microarray determination of Bcl-2 family protein inhibition sensitivity in breast cancer cells.

This study tests the hypothesis that reverse transcription polymerase chain reaction (RT-PCR) microarrays can be used to predict the relative sensitivity to induction of apoptosis in breast cancer cells exposed to inhibitors of antiapoptotic Bcl-2 family proteins. Four cell lines, MDA-MB-231 (MDA-231) and MDA-MB-468 (MDA-468), BT-20 and T47-D were screened for relative expression of Bcl-2 family members A1, Mcl-1, Bcl-2, Bcl-xL and Bcl-w mRNA by RT-PCR microarrays and Western analysis. The four cell lines were treated with 1 μmol/L obatoclax (GX15-070) and/or 2 Gy radiation (RT) and monitored for apoptosis after 48 h.

WR-1065, the active form of amifostine, protects HL-60 cells but not peripheral blood mononuclear cells from radiation and etoposide-induced apoptosis.

Purpose: Developing myeloid cells are particularly sensitive to chemotherapy and ionizing radiation. Mature cells of the hematopoietic lineages, such as are found in the peripheral blood mononuclear cells (PBMCs), are much less sensitive for reasons that are not yet understood. Protecting the myeloid precursors from radiation or chemotherapy is an important goal.

Bcl2-independent chromatin cleavage is a very early event during induction of apoptosis in mouse thymocytes after treatment with either dexamethasone or ionizing radiation.

We have quantified the emergence of early chromatin breaks during the signal transduction phase of apoptosis in mouse thymocytes after treatment with either ionizing radiation or dexamethasone. Dexamethasone at 1 microM can induce significant levels of DNA breaks (equivalent to the amount induced directly by 7.5 Gy ionizing radiation) within 0.

Differential sensitivity of double minute chromosomes to hydroxyurea treatment in cultured methotrexate-resistant mouse cells.

Treating mammalian cells with continuous sub-lethal doses of Hydroxyurea (HU) causes the loss of double minute chromosomes (DMs) containing amplified oncogenes in culture. Recently, we have shown that treating glioblastoma multiforme cells in culture with low doses of HU causes the loss of DMs containing epidermal growth factor receptor genes. Loss of amplified EGFR genes was accompanied by cessation of growth, and greatly decreased tumorigenicity.

No detectable misrejoining in double-minute chromosomes.

Pulsed-field gel electrophoresis combined with Southern hybridization and rare-cutting restriction endonuclease digestion has been used recently to quantify misrejoining of DNA double-strand breaks (DSBs) resulting from exposure to ionizing radiation. Measurements are made 24 h after a high dose of radiation. These studies have suggested that a large fraction of DSBs are misrejoined to result in gross rearrangements.

The scid defect results in much slower repair of DNA double-strand breaks but not high levels of residual breaks.

Severe combined immune deficiency (scid) mice fail to produce mature B and T cells and are sensitive to ionizing radiation. They contain a mutation in the 460-kDa catalytic subunit of the DNA-dependent protein kinase that is involved in both V(D)J rejoining and DNA double-strand break (DSB) repair. The kinetics of DSB rejoining was quantified in both scid cells and the parental C.

An assay for quantifying DNA double-strand break repair that is suitable for small numbers of unlabeled cells.

A system based on pulsed-field gel electrophoresis (PFGE) is described which measures the induction and repair of DNA double-strand breaks (DSBs) in a biologically relevant X-ray dose range (below 10 Gy) using as few as 125 cells per time. This system was used to measure repair in cells of a freshly obtained human glioblastoma multiforme tumor. No prelabeling of the cells is required, and many different cell types can be studied using this system.

Hydroxyurea accelerates the loss of epidermal growth factor receptor genes amplified as double-minute chromosomes in human glioblastoma multiforme.

Objective: We sought to determine whether hydroxyurea could accelerate the loss of amplified epidermal growth factor receptor (EGFR) genes from glioblastoma multiforme (GBM). There is good reason to think that elimination of amplified EGFR genes from GBMs will negatively impact tumor growth. Hydroxyurea has previously been shown to induce the loss of amplified genes from extrachromosomal double minutes (dmin) but not from chromosomal homogeneously staining regions.

Induction and repair of DNA double-strand breaks in the same dose range as the shoulder of the survival curve.

We have used pulsed-field gel electrophoresis (PFGE) to test two hypotheses that have been proposed to explain the survival curves with shoulders which are characteristic of low-LET ionizing radiation: (1) Neutral elution studies of the induction of double-strand breaks (DSBs) have suggested that ionizing radiation might induce DSBs in a nonlinear fashion at low doses. (2) Based on analogies to enzyme kinetics, DSB repair might be saturating in the shoulder region. We quantified DSB induction and survival resulting from doses between 0 and 5 Gy spanning the shoulder region of the survival curve.

Hyperthermia inhibits the repair of DNA double-strand breaks induced by ionizing radiation as determined by pulsed-field gel electrophoresis.

Hyperthermia is known to synergistically interact with X-rays to kill cells. We have used pulsed-field gel electrophoresis to investigate the effects of hyperthermia on cell survival and on repair of radiation-induced DNA double-strand breaks (dsbs). Combining hyperthermia (43 degrees C, 45 min) with radiation (7.

Induction and repair of DNA double-strand breaks.

Induction and repair of DNA double-strand breaks (DSBs) was measured using a pulsed-field gel electrophoresis system. A cell line of methotrexate-resistant EMT-6 cells that contain numerous double-minutes (DMs) 3 million base pairs in size was employed. The electrophoretic mobility of these DMs depends on whether they have zero, one, or more than one DSB.

Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe, to detect submicroscopic DMs. One of the cloned MTX-resistant isolates was examined in detail and was shown to originally contain amplified DHFR genes on circular DMs measuring 1 and 3 Mb in size; additionally, metaphase chromosome preparations from this cloned isolate were examined and were shown to contain microscopically visible DMs too large to enter a pulsed-field gel.

Direct measurement by pulsed-field gel electrophoresis of induction and rejoining of X-ray-induced double-strand breaks in cultured mouse cells.

The induction and rejoining of X-ray-induced double-strand breaks (dsb) in chromosomal DNA has been difficult to measure. We have developed a pulsed-field gel electrophoresis (PFGE)-based system for directly estimating DNA sizes between 0.2 and 10 million base pairs.

X-ray induction of methotrexate resistance due to dhfr gene amplification.

The effect of ionizing radiation on methotrexate (MTX) resistance and gene amplification in cultured mammalian cells was investigated. X-irradiation of mouse EMT-6 cells induced cell killing and MTX resistance due to amplification of dihydrofolate reductase (dhfr) gene in a dose-dependent manner. The highest yields of mutant cells were obtained at approximately D37 (the dose at which 37% of the cells survive), where the frequency of MTX-resistant cells was four- to eightfold over that of the unirradiated population.

Acyl-coenzyme A carboxylase of the free-living nematode Turbatrix aceti. 1. Its isolation and molecular characteristics.

A biotin containing enzyme which carboxylates acetyl-CoA has been isolated from the nematode Turbatrix aceti and purified to homogeneity as judged by the criteria of polyacrylamide gel electrophoresis and ultracentrifugation. The enzyme has a sedimentation coefficient of 18.0 S and a molecular weight of 667 000.