High-resolution analysis and purification of synthetic oligonucleotides with strong anion-exchange HPLC.

A strong anion-exchange high-performance liquid chromatography column, the NucleoPac PA-100, has been developed for high-resolution analysis and purification of synthetic oligonucleotides. Very high-resolution separations of oligonucleotides to 60 bases can be obtained. Separations can be performed under non-denaturing or denaturing conditions because the column can be operated routinely at high temperature or high pH. Analytical methods can be directly transferred to larger NucleoPac PA-100 columns for semi-preparative applications. In this report, methods for the analysis of synthetic oligonucleotides under non-denaturing (pH 8.0) and denaturing (pH 12.4) conditions are presented. A gradient separation for machine-grade DNA at pH 8.0 is developed. Transfer of an analytical method to a 22-mm-diameter semi-preparative column is also demonstrated.

Adaptation of EGF receptor signal transduction to three-dimensional culture conditions: changes in surface receptor expression and protein tyrosine phosphorylation.

A431 cells grown as three-dimensional spheroids show growth stimulation in response to nanomolar concentrations of EGF in contrast to monolayer cultures that show inhibition. In investigating the alterations in EGF signal transduction that underlie this modification of the proliferative response, we have compared the expression of EGF receptors on A431 cells under these conditions and related our findings to tyrosine phosphorylation and the growth response. EGF receptors were measured by 125I-EGF binding to trypsin-dispersed cells. Unexpectedly, dispersion of the monolayers caused an 80% decrease in surface EGF receptor, although, after dispersion, EGF receptor was digested by trypsin with a half-life of 69 +/- 32 min. No evidence for a comparable loss of cellular EGF receptor was seen on trypsin dispersion of spheroids. After allowing for this effect, we found that the receptor density on nondispersed monolayers (5 x 10(6) per cell) was twentyfold greater than that on spheroids (0.25 x 10(6) per cell). EGF-induced tyrosine phosphorylation was confined to the outermost cells of the spheroid, although the presence of surface-expressed EGF binding sites could be demonstrated throughout the structure and the number of EGF receptors/cell on dispersed spheroid cells showed a single distribution peak by flow cytometry, with no evidence for more than one population. Using RCM-lysozyme as a substrate, tyrosine phosphatase activity in spheroids lay within the range observed in monolayer cultures. Autophosphorylation of the EGF receptor following EGF stimulation in monolayer cultures of A431 cells rose rapidly in the first 10 seconds and then slowly increased for at least 3 h. In spheroids, it reached a maximum within 10 seconds and then declined over 3 h. Since the microenvironment within a tumor resembles that in a spheroid, a similar reduction in surface EGF receptor expression may be expected in tumors relative to monolayer cultures, together with corresponding growth stimulation in response to EGF.

Regulation of c-jun expression during hypoxic and low-glucose stress.

Hypoxic stress in tumor cells has been implicated in malignant progression and in the development of therapeutic resistance. We have investigated the effects of acute hypoxic exposure on regulation of the proto-oncogene c-jun in SiHa cells, a human squamous carcinoma cell line. Hypoxic exposure produced increased levels of c-jun mRNA resulting from both message stabilization and transcriptional activation. A superinduction of c-jun message resulted during simultaneous oxygen and glucose deprivation, with several characteristics of an induction mediated by oxidative-stress pathways. This superinduction was blocked by preincubation of cells with the glutathione precursor N-acetyl cysteine or with phorbol 12-myristate 13-acetate, which indicates redox control of c-jun expression and probable involvement of protein kinase C. By gel retardation assay, no increase in AP-1 DNA binding activity was found to be concomitant with the transcriptional activation of c-jun. A lack of increased DNA binding was observed for the consensus AP-1 sequence and for the two AP-1 sequence variants found within the c-Jun promoter. Additionally, hypoxic and low-glucose stress produced no activation of stably transfected AP-1 reporter sequences. Taken together, these results indicate that the transcriptional activation of c-jun during hypoxic and low-glucose stress involves redox control and is unlikely to be mediated by AP-1 recognition elements within the c-jun promoter.

Epidermal growth factor modifies cell cycle control in A431 human squamous carcinoma cells damaged by ionizing radiation.

Epidermal growth factor (EGF) has been shown to radiosensitize A431 and other human squamous carcinoma cells with high numbers of surface EGF receptors. In this study of the mechanistic basis of EGF-induced radiosensitization, both EGF and ionizing radiation caused G1 phase arrests in cycling A431 cells, but only radiation caused a G2-M arrest. However, EGF enhanced the magnitude of this G2-M arrest, suggesting an interaction of signaling pathways involved in cellular responses to EGF and radiation damage. EGF and radiation also uniquely perturbed cyclin A and B1 mRNA levels during the time of maximum radiation-induced G2-M arrest. The effects of EGF on G2-M events probably originated in cells in G1. It is possible that aberrant EGF signal transduction in human squamous carcinoma cells may be exploited as a novel strategy for radiotherapy.

Quantitative imaging of a radiotherapeutic drug, Na2B12H11SH, at subcellular resolution in tissue cultures using ion microscopy.

The effectiveness of boron neutron capture therapy is predicted to be dependent not only on the amount of boron taken up by the target cells but also on the intracellular distribution of boron. Using the isotopic imaging technique ion microscopy, we have quantitatively determined uptake and intracellular distribution of Na2B12H11SH, a promising boron drug for boron neutron capture therapy, in four human cell lines: U87 glioblastoma cells, HeLa epithelioid carcinoma cells, GM 2408b mutant skin fibroblasts, and GM 3348b skin fibroblasts. The boron uptake of all four cell lines, after exposure to 100-500 micrograms/ml Na2B12H11SH, increased as the dosages were increased but showed a tendency toward saturation. Boron was more concentrated in the cytoplasm than in the nucleus but was not strongly localized within cells. There were no significant differences in boron uptake among the four cell lines. A retention experiment identified at least two different intracellular boron pools, and cells lost greater than 60% of intracellular boron within 1 h upon changing to Na2B12H11SH-free medium, indicating a largely low affinity binding.

Subcellular imaging of calcium exchange in cultured cells with ion microscopy.

Calcium to calcium exchange between intracellular and extracellular pools has been imaged directly in individual cells using stable 44Ca (98.78% enrichment) in the nutrient medium and an isotopic imaging technique, ion microscopy. Observations were made by imaging mass 40 to determine the native intracellular calcium (40Ca), and mass 44 to localize the 44Ca from the extracellular medium that exchanged with cellular calcium. LLC-PK1 porcine kidney epithelial cells were exposed to the nutrient medium with 1.87 mM stable 44Ca for 0, 1, 2, 5, 8, 20, 60 and 90 min, and cryogenically prepared prior to ion microscopic analysis. The cell nucleus, the Golgi region and the remaining cell cytoplasm could be spatially resolved to within about 0.5 microns using the ion microscope. On the basis of the kinetics of 40Ca to 44Ca exchange it was observed that all three compartments had a rapidly exchanging pool of calcium, which took about 2 min to exchange. A moderately rapidly exchanging pool of calcium was identified between 2 and 20 min of calcium exchange. The cells had exchanged about 50% of their total internal calcium with the external calcium in less than 20 min. The remaining 50% of the cellular calcium could be classified as a slowly exchanging pool. Isotopic images of 39K and 23Na were recorded along with 40Ca and 44Ca images to assess the health status of the cell. Isotopic imaging has the unique ability to distinguish intracellular calcium from the extracellular calcium that enters cells and has enormous potential for studies of calcium transport under physiological and pathological conditions.

Quantitative imaging of boron, calcium, magnesium, potassium, and sodium distributions in cultured cells with ion microscopy.

A method for the conversion of intensity information in ion micrographs of freeze-fractured, freeze-dried cultured cells to local dry weight elemental concentrations is presented. Homogenates generated from cultured cells are used as calibration standards. Ion microscope (IM) relative sensitivity factors for B, Ca, K, Mg, and Na with respect to the matrix element C are determined by the correlation of IM and inductively coupled plasma atomic emission spectrometry analyses of the cellular homogenates. After calibration of the IM imaging system, the relative sensitivity factors are used to determine local intracellular concentrations of B, Ca, K, Mg, and Na in cultured Swiss 3T3 fibroblasts. Intracellular B was introduced through cellular uptake of Na2B12H11SH, a candidate therapeutic agent for boron neutron capture cancer therapy. The IM intracellular concentration results show good agreement with published electron probe X-ray microanalysis results. Estimated detection limits are in the low- to subparts-per-million dry weight concentration range.

Morphological and elemental integrity of freeze-fractured, freeze-dried cultured cells during ion microscopic analysis.

The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. 41K+/24Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of 39K+, 23Na+, 40Ca+ and 24Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some 24Mg+ micrographs. Various non-uniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.

Evaluation of matrix effects in ion microscopic analysis of freeze-fractured, freeze-dried cultured cells.

SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze-fractured, freeze-dried cultured cells at the approximately 0.5 micron spatial resolution of the Cameca IMS-3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H-, C-, O-, Na+, Mg+, CN-, P-, S-, Cl-, K+ and Ca+ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distributions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.