Preferential enlargement of leukemia cells using cytoskeletal-directed agents and cell cycle growth control parameters to induce sensitivity to low frequency ultrasound.

Sonodynamic therapy (SDT) is a form of ultrasound therapy that has been shown to preferentially damage malignant cells based on the relatively enlarged size and altered cytology of neoplastic cells in comparison to normal cells. This study sought to determine whether cytoskeletal-directed agents that either disrupt (cytochalasin B and vincristine) or rigidify (jasplakinolide and paclitaxel) microfilaments and microtubules, respectively, affect ultrasonic sensitivity. U937 human monocytic leukemia cell populations were treated with each cytoskeletal-directed agent alone, and then sonicated at 23.5 kHz under relatively low power and intensity (20-40 W; 10-20 W/cm(2)), or at 20 kHz using moderate power and intensity (60 W; 80 W/cm(2)). In addition, human leukemia lines U937, THP1, K562, and Molt-4, and the murine leukemia line L1210 were sonicated using pulsed 20 kHz ultrasound (80.6 W; 107.5 W/cm(2)) both with and without the addition of cytoskeletal-directed agents to assess whether cytoskeletal-directed agents can potentiate ultrasonic sensitivity in different leukemia lines. Human hematopoietic stem cells (hHSCs) and leukocytes were sonicated with continuous 23.5 kHz ultrasound (20 W; 10 W/cm(2)) to determine whether this approach elicited the preferential damage of neoplastic cells over normal blood components. To determine whether ultrasonic sensitivity is exclusively dependent on cell size, leukemia cells were also enlarged via alteration of cell growth parameters including serum deprivation and re-addition, and plateau-phase subculturing. Results indicated that cytochalasin B/ultrasound treatments had the highest rates of initial U937 cell damage. The cells enlarged and partially synchronized, either by serum deprivation and re-addition or by plateau-phase subculturing and synchronous release, were not comparably sensitive to ultrasonic destruction based solely on their cell size. In addition, cytochalasin B significantly potentiated the ultrasonic sensitivity of all neoplastic cell lines, but not in normal blood cells, suggesting that preferential damage is attainable with this treatment protocol. Therefore, it is likely that ultrasonic cell lysis depends not only on cell size and type, but also on the specific molecular mechanisms used to induce cell enlargement and their effects on cell integrity. This is supported by the fact that either the microfilament-or microtubule-disrupting agent produced a higher rate of lysis for cells of a given size than the corresponding stabilizing agents.

Targeting aldehyde dehydrogenase: a potential approach for cell labeling.

INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

64Cu-azabicyclo[3.2.2]nonane thiosemicarbazone complexes: radiopharmaceuticals for PET of topoisomerase II expression in tumors.

UNLABELLED: Topoisomerase II (Topo-II) is an essential enzyme in the DNA replication process and is the primary cellular target for many of the most widely used and effective anticancer agents. It has been reported that thiosemicarbazones (TSCs) are potent antitumor agents that inhibit Topo-II. The aim of this study was to investigate the relationship between the in vitro and in vivo behavior of novel (64)Cu-TSC complexes and the expression of Topo-II activity. METHODS: Four (4)N-azabicyclo[3.2.2]nonane TSC derivatives (EPH142, EPH143, EPH144, and EPH270) were successfully radiolabeled with (64)Cu, to form lipophilic cations of the general formula [(64)Cu(L)]Cl, and the partition coefficient (logP) values were determined. One agent [(64)Cu-EPH270](+) was observed in vitro in cultured cell studies. The kinetics of 2 compounds, [(64)Cu-EPH144](+) and [(64)Cu-EPH270](+), were examined in mice bearing L1210 tumors and small-animal PET was conducted in mice bearing L1210 and PC-3 tumors, which expressed high and low levels of Topo-II, respectively. All data were compared with the activity and levels of Topo-II, as determined by a commercially available assay kit and western blot analysis. RESULTS: The 4 complexes were radiolabeled by incubation of (64)CuCl(2) with the ligand in ethanolic solution. The complexes were isolated in high radiochemical purity, as determined by radio-thin-layer chromatography and radio-high-performance liquid chromatography. The compounds were shown to be lipophilic with logP values ranging from 1.34 to 1.92. In biodistribution studies, good L1210 tumor uptake was noted ([(64)Cu-EPH144](+) at 1 h, 4.70 %ID/g [percentage injected dose per gram]; 4 h, 8.80 %ID/g; 24 h, 6.64 %ID/g; and [(64)Cu-EPH270](+) at 1 h, 2.58 %ID/g; 4 h, 6.00 %ID/g; 24 h, 4.80 %ID/g). Small-animal PET of animals with L1210 tumors (high Topo-II expressing) showed excellent tumor accumulation compared with that of animals with PC-3 tumors (low Topo-II expressing), and the L1210 tumor uptake was significantly reduced by coadministration of a Topo-II poison. CONCLUSION: Here we describe the characterization of a new class of copper-radiolabeled TSC analogs. We demonstrate that the accumulation of the (64)Cu-compounds is related to the expression levels of Topo-II in tumor tissue.

Scintigraphic detection in mice of inflammatory lesions and tumours by an indium-labelled monoclonal antibody directed against Mac-1 antigen.

The monoclonal antibody, 3A33, directed against Mac-1 antigen which is expressed essentially on macrophages and polymorphonuclear cells, was injected i.v. into mice, as part of an attempt to visualize inflammatory lesions and tumours by external scintigraphy. The monoclonal antibody, a rat IgG2a, was conjugated with a bifunctional chelating agent, diethylenetriaminepentaacetic acid at a 1:1 molecular ratio and complexed with 111-indium, a procedure which apparently did not alter its binding to peritoneal macrophages and provided relatively stable cell labelling. An unrelated rat IgG2a of unknown specificity radiolabelled in the same manner as 3A33 served as a control. The uptake of i.v. injected 3A33 by peritoneal macrophages was up to 50 times that of unrelated IgG2a. After i.v. inoculation, the antibody accumulated in the liver, spleen, lung, in foot-pad inflammatory reactions induced by injection of Freund's adjuvant and in experimentally grafted tumours. The 3A33: non-specific IgG2a uptake ratio in inflammatory lesions and tumours, however, was much lower than for peritoneal macrophages and was generally close to 2. This was sufficient to obtain scintigraphic images of inflammations and tumours. The images obtained after injection of 3A33 were clearly of better quality than those given by the non-specific immunoglobulin. They could be improved by subtraction of the vascular images obtained after injection of 99m-technetium serum albumin. The labelling of Mac-1-positive blood mononuclear cells by in vitro incubation with radioactive 3A33 was not intense enough to allow scintigraphic imaging after in vivo re-infusion but seemed more selective than the injection of whole antibody in detecting inflammatory reactions. These results seem interesting in view of the potential human application to the detection inflammatory lesions and the appreciation of tumour inflammatory components. Possible improvements in the technique are discussed.

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice.

In vivo localization of a mouse monoclonal antibody (F2-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab')2 fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radio-localization studies were performed by using 125I- or 131I-labeled IgM monoclonal antibody or its F(ab')2 fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab')2 fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of 125I-radiolabeled F(ab')2 fragments and 125I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab')2 fragments was lower than with IgM, and so gamma-camera imaging was workable with F(ab')2 fragments without background subtraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab')2 fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab')2 fragments make them hardly suitable as carriers of toxic drugs.