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.

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.

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SUMMARY SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze‐fractured, freeze‐dried cultured cells at the ∼0.5 μm 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 distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.