Noninvasive imaging of c-myc oncogene messenger RNA with indium-111-antisense probes in a mammary tumor-bearing mouse model.

UNLABELLED: The c-myc oncogene is amplified in leukemia and solid tumors, thus making the c-myc messenger RNA (mRNA) a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense pharmaceuticals or radiolabeled antisense oligodeoxynucleotide (RASON) probes. Considering the higher stability of phosphorothioate over phosphodiester, the probe stability and tumor localization was compared with both derivatives. METHODS: The 15-mer oligonucleotide sequence was synthesized, aminolinked [sense and antisense phosphodiester (O) and monothioester (S)] and coupled with diethylenetriamine pentaacetate (DTPA)-isothiocyanate and aliquots were lyophilized to make a DTPAAHON kit. The radionuclide 111In was chelated to DTPAAHON derivatives, and free 111In was separated by gel filtration. The radiolabeled antisense and sense probes were injected intravenously in mammary tumor-bearing BALB/c mice (1 x 10(6) cells, 8 days postinoculation). RESULTS: The highest uptake was observed at 2 hr with both thio and oxo derivatives of RASON probes, and small tumors could be imaged noninvasively. Tumor uptake and tumor/blood and tumor/muscle ratios for the sense probe (control) were significantly lower (p < 0.001) than those of the antisense probe. CONCLUSION: The radiolabeled antisense probe may provide a new sensitive tool for noninvasive imaging of c-myc oncogene mRNA for a variety of malignant tumors at an earlier stage.

Kinetics of hybridization of mRNA of c-myc oncogene with 111In-labeled antisense oligodeoxynucleotide probes by high-pressure liquid chromatography.

Antisense oligodeoxynucleotides (ASON) were labeled with gamma-emitting 123I, 99mTc and 111In radionuclides. The hybridization kinetics of 111In-labeled ASON probes [phosphorothioate (S) and phosphodiester (O)] with intact leukemic cells (P388) and purified mRNA was studied by gel filtration technique. The 15-mer oligodeoxynucleotide (ON) sequence was synthesized, amino linked and coupled to diethylenetriaminepentaacetic acid (DTPA)-isothiocyanate, and aliquots were lyophilized to make a kit for convenient preparation. 111In radionuclide was chelated to DTPAASON derivatives and free 111In was separated by gel filtration. The probe was incubated with P388 cells and mRNA extract of P388 cells. Hybridization kinetics was studied by measuring the free and mRNA-bound probe separated by the HPLC technique. The distribution of radioactivity associated with proteins, DNA and mRNAs was directly measured with a gamma camera and further quantified with an ionization chamber. The kinetics of direct and indirect hybridization of 111In-labeled antisense probes with mRNA and intact cells was similar.

A higher blood flow window of reduced thrombogenicity and acceptable fragmentation in a hollow fiber hemodialyzer.

The effect of pulsatile blood flow on platelet thrombogenicity and platelet fragmentation (PF) in a hollow fiber hemodialyzer (HFD) was quantified with 111In labeled platelets and 125I labeled fibrinogen; 150 ml of blood was collected from Beagle dogs, Yorkshire pigs, and a human volunteer (non-smoker). Platelets were labeled with 111In tropolone (300 microCi) and fibrinogen was labeled with 125I. Sham dialysis (SHD) was performed with 120 HFDs (0.9 meter2) at 37 degrees C, with flow-rates of 150, 250, 500, and 950 ml/min.; after SHD, the washed HD radioactivity was measured with an ionization chamber. PF was measured by flow cytometry with GP IIb-IIIa murine monoclonal antibody. Platelet deposition decreased significantly for 3 species at higher flow; fibrinogen deposition (10-12%, 55-65 mg/m2), was not affected by flow. Adherent platelet thrombus decreased from (8.2 +/- 3.4) to (3.1 +/- 1.0) with human blood as flow rate increased from 150 to 950 ml/min; platelet thrombus level also decreased significantly (p < 0.005) from (20.3 +/- 6.2) to (4.5 +/- 1.9) with canine blood. Higher values were obtained for canine than human and porcine platelets. Platelet fragmentation, on the other hand, increased from 2.1-2.2% to 10.2-11.3% with increase of flow. Like platelets, deposition of canine fibrinogen was slightly higher than that of pig and human. The studies of adherent thrombus and platelet fragmentation identified an important flow-window of reduced thrombogenicity and acceptable fragmentation, encouraging extracorporeal circulation at higher blood flow.

Development of sensitive radioiodinated anti-sense oligonucleotide probes by conjugation technique.

Radioactive and nonradioactive oligonucleotide (ON) probes have been used in a variety of studies of in vitro hybridization for locations of specific genes and determination of the level of mRNA transcription in activated and proliferative cells. Nuclease-resistant phosphorothioate derivatives of oligonucleotides are also finding potential therapeutic applications in intact cells and animal models. In this study, we have developed new techniques of radioiodination by the conjugation of oligonucleotides with p-methoxyphenyl isothiocyanate (PMPITC) and optimized the conditions of the radioiodination, e.g. concentration of reactants, oxidants, pH, time, and temperature. We have used a 25-mer actin mRNA probe as a model substrate for radioiodination. The ON probe was modified by conjugation with the addition of aminohexyl (AH) group at the 5'-terminal. Both the techniques of preiodination of PMPITC followed by conjugation and radioiodination of PMPITC-conjugated AHON were evaluated; the latter was found to be more convenient. The optimum pH of radioiodination of PMPITC with 125I was 6.5; on the other hand, the optimum pH for PMPITC-conjugated AHON was 8.5. The conditions of conjugation were optimized with 125I-labeled PMPITC. The techniques of paper chromatography and HPLC with a Spherogel column were developed for the purification and characterization of these oligonucleotide probes. Without PMPITC conjugation, the yield of direct radioiodination of the same oligonucleotide was very low (4-6%); the yield in our optimized technique is 50-60%. Radioiodination of conjugated oligonucleotides is a simple and efficient route to radiolabeled probes for hybridization studies.