We demonstrate here that functional NMDAR1 receptors and NMDAR2 receptors are expressed by Mcf-7 and SKBR3 breast cancer cell lines, and possibly by most or all high-grade breast tumors, and that these receptors are important for the growth of human breast cancer xenografts in mice. RT-PCR demonstrated mRNA for both NMDAR1 and NMDAR2 receptors are expressed in both Mcf-7 and SKBR3 cell lines, and these messages likely have sequences identical to those reported for human mRNAs. Proteins of the expected respective sizes 120 and 170 KD are generated from these mRNAs by the tumor cells. Cell growth was found to be significantly (p<0.0001) impaired down to 10% of normal growth by the irreversible NMDAR1 antagonists MK-801 and memantine with IC 50s ranging from 600–>800 μM and from 200–300 μM for the two lines. Paradoxically, memantine with a lower binding affinity had the greater influence of the two inhibitors on cell viability. Immunohistochemical examination of high-grade invasive ductal and lobular breast cancer with our polyclonal antibodies against a peptide (-Met-Ser-Ile-Tyr-Ser-Asp-Lys-Ser-Ile-His-) in the extracellular domain of the NMDAR1 receptor gave specific positive staining for the receptor in all 10 cases examined. Positive staining was chiefly concentrated at the membranes of these tumor tissues. No staining with these antibodies was found for normal breast and kidney tissues. When Mcf-7 cells were grown as tumor xenografts in nu/nu mice, the growth of these tumors was completely arrested by daily treatments with MK-801 over five days. All of these data point to active NMDAR receptors being expressed by most breast cancers, and having an important influence on their survival.

Human y (immune) interferon (IFN-y) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-yis a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 ± 3000. A purification process for IFN-y was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 104 (crude culture fluid) to an estimated 107 units per mg of protein with a cumulative recovery of about 40% of the IFN activity.

No abstract

Early fibrinolytic therapy with full molecular tissue plasminogen activator (t-PA) has been observed to be both angiographically and clinically effective when employed in animal stroke models. Preliminary clinical trials with t-PA are in progress. It is possible to refine t-PA by developing fragments or analogues of the drug. Using recombinant DNA technology in the Escherichia coli system, a t-PA analogue consisting of the catalytic fragment of t-PA and a dimer of the B fragment of staphylococcal protein A (Fb-Fb-CF) has been produced. Because this analogue has a long serum half-life of 90 minutes, we employed Fb-Fb-CF in a rabbit cerebral embolic stroke model to assess its efficacy as a reperfusion agent. When given as a bolus to 10 animals 15 minutes after embolization, Fb-Fb-CF produced angiographic cerebral reperfusion in 48 +/- 21 minutes (+/- SD), while in 8 saline-treated controls, reperfusion was not observed at 180 minutes in any animal (p less than 0.01). In another experiment reperfusion was demonstrated at 66 +/- 32 minutes in 11 animals treated with Fb-Fb-CF 90 minutes after embolization as compared with 100 +/- 25 minutes in 12 saline-treated controls (p less than 0.01). A small macroscopic hemorrhage within an infarct was seen in 1 Fb-Fb-CF-treated animal in the 15-minute experiment and in none of the controls. In the 90-minute experiment, macroscopic hemorrhagic infarction was seen in 4 Fb-Fb-CF-treated animals and in 3 controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.