Comparative evaluation of fresh, fixed, and cryopreserved solid tumor cells for reliable flow cytometry of DNA and tumor associated antigen.

Five different protocols for the short-term preservation of cells used for multiparameter flow cytometric assay of tumour associated antigens (TAA) and DNA were assessed in cell suspensions prepared by mechanical disaggregation of 15 gynecological tumors. The protocols at 4 degrees C were 1) storage in buffer, 2) storage in 50% methanol, and 3) storage in buffer after formalin fixation. Tissues were also cryopreserved as cell suspensions and tissue blocks. When the TAA expression and DNA histograms of the preserved cells were compared with those in fresh cell suspensions, cryopreservation was found to be the best method: TAA expression was well preserved and there was a good correlation between TAA expression and the quality of the DNA histograms, respectively, in fresh and cryopreserved cells (RS: 0.82-0.91, P less than 0.001 for all TAAs). The cell suspensions preserved at 4 degrees C all showed a significant increase in background fluorescence (P less than 0.05) and a reduction in the TAA specific fluorescence (P less than 0.011). Methanol fixation was better than buffered formalin for the proteins studied, though both gave significantly worse results than cryopreservation. The quality of these cell suspensions and the correlation with TAA measurements in fresh cell suspensions deteriorated progressively with time, particularly if they were stored more than a week.     

Eicosanoids in breast cancer patients before and after mastectomy.

In 19 patients with a malignant breast tumor, tumor tissue and blood were taken to determine the eicosanoid profile and platelet aggregation. Values were compared with those of patients with benign tumors (n = 4), or undergoing a mammary reduction (n = 7). Postoperatively, blood was taken as well in order to compare pre- and postoperative values. Eicosanoids were measured in peripheral blood monocytes and mammary tissue by means of HPLC; furthermore, TXA2, 6-keto-PGF1 alpha, and PGE2 were determined by RIA. Differences in pre- and postoperative values of cancer patients were seen in plasma RIA values: PGE2 and 6-k-PGF1 alpha were significantly higher preoperatively when compared with postoperatively, however, such differences were seen in the control groups as well. Compared to benign tumor or mammary reduction test material the eicosanoid profile of tissue obtained from malignant mammary tumors showed important differences. Except for PGF2 alpha, HHT and 15-HETE no detectable quantities of eicosanoids were found in the non-tumor material, whereas in the malignant tumor material substantial quantities of a number of eicosanoid metabolites were present. Statistically significant correlations could be established between patient/histopathology data and the results of the platelet aggregation assays, e.g. between menopausal status and ADP aggregation; oestrogen receptor (+/-) and collagen and arachidonic acid aggregation, inflammatory cell infiltration score and arachidonic acid aggregation and fibrosis score and ADP aggregation. The results show that eicosanoid synthesis in material from mammary cancer patients is different from that in benign mammary tissue. The implications, in particular, in relation to future prognosis of the patient, remain obscure.     

Air pollution as a possible cause for the decline of some phanerogamic species in The Netherlands

In The Netherlands the decline of some phanerogamic species cannot be readily explained from obvious factors such as lowering of the groundwater table, eutrophication or land reclamation. For a number of species the hypothesis was tested that the decline is partly due to air pollution. A two-factor model was made in which decline is accounted for by (a) habitat destruction assessed from topographic maps and (b) air pollution measured as the SO₂ 95-percentile over the winter period 1978/1979. Effects of both factors were assumed to follow a sigmoid dose-effect curve. For a number of species decline proved to be significantly correlated with air pollution. These are notably species from the syntaxon Violion caninae. A comparison was made with results obtained for epiphytic lichens. It appears that for some phanerogamic species sensitivity is about the same as for moderately sensitive lichens.Nomenclature follows Heukels & van der Meijden (1983).Thanks are due to the Rijksherbarium, for providing some of their unpublished data; and to Ada Groeneveld, for technical assistance.     

Flow cytometric detection of multifocal DNA aneuploid cell populations in mastectomy specimens containing a primary breast carcinoma

DNA flow cytometry was used to study the presence of DNA aneuploid cell populations in macroscopically normal glandular tissue in mastectomy specimens from 30 patients with breast cancer. In the 13 patients with a DNA diploid primary tumor, no DNA aneuploidy could be found in any of the 39 distant specimens assessed. However, DNA aneuploid cell populations were demonstrated in four of the 17 (23%) patients with a primary DNA aneuploid carcinoma and in seven out of 54 (13%) distant tissue samples (P = 0.02). In all cases the DNA index of the DNA aneuploid cells found in the distant samples was identical to that of the primary tumor. The replicate aneuploid DNA indices and histologic controls taken in parallel very strongly suggest that these distant DNA aneuploid cell populations are metastases.     

The CR1 and CR3 domains of the adenovirus type 5 E1A proteins can independently mediate activation of ATF-2.

The adenovirus 12S E1A protein can stimulate the activity of the c-jun promoter through a conserved region 1 (CR1)-dependent mechanism. The effect is mediated by two AP-1/ATF-like elements, jun1 and jun2, that preferentially bind c-Jun-ATF-2 heterodimers. In this study, we show that the ATF-2 component of the c-Jun-ATF-2 heterodimer is the primary target for 12S E1A: 12S E1A can enhance the transactivating activity of the N terminus of ATF-2 when fused to a heterologous DNA-binding domain, whereas the transactivating activity of the c-Jun N terminus is not significantly affected. Activation of the ATF-2 N terminus by 12S E1A is dependent on CR1. In the context of the 13S E1A protein, CR1 and CR3 can both contribute to activation of ATF-2, and their relative contributions are dependent on the cell type. In contrast to activation of ATF-2 by stress-inducing agents, CR1-dependent activation of ATF-2 was found not to depend strictly on the presence of threonines 69 and 71 in the N terminus of ATF-2, which are targets for phosphorylation by stress-activated protein kinases (SAPKs). In agreement with this observation, we did not observe phosphorylation of threonines 69 and 71 or constitutively enhanced SAPK activity in E1A- plus E1B-transformed cell lines. These data suggest that CR1-dependent activation of ATF-2 by 12S E1A does not require phosphorylation of threonines 69 and 71 by SAPK.     

Role of glucose signaling in yeast metabolism

In this article, knowledge concerning the relation between uptake of and signaling by glucose in the yeast Saccharomyces cerevisiae is reviewed and compared to the analogous process in prokaryotes. It is concluded that (much) more fundamental knowledge concerning these processes is required before rational redesign of metabolic fluxes from glucose in yeast can be achieved. (c) 1996 John Wiley & Sons, Inc.