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Background: Topical corticosteroids are the only effective measure in serious inflammatory corneal and conjunctival diseases. Although results obtained with topical cyclosporin A are encuraging it is not effective in all patients. The mode of action of Fk506 is similar to that of cyclosporin A, i.e. it exerts an inhibitory effect on transcription of interleukin 2 in T lymphocytes. The immunosuppressive potential of Fk506, however, is much larger. Furthermore, it penetrates more easily into cornea and conjunctiva. To find out whether the theoretical advantages of topical Fk506 can be translated into clinical practice, a selected group of patients refractory to conventional therapy was treated in this pilot study., Patients and Methods: Fk506 0.06 % was administered initially three times daily in 15 patients with atopic blepharokeratoconjunctivitis, Mooren's ulcer, ocular pemphigoid, Thygeson's superficial punctate keratitis, nummular adenoviral keratitis, graft-versus-host reaction of the conjunctiva and steroid response glaucoma after penetrating keratoplasty., Results: Within a follow-up of 26 +/- 15 weeks improvement was recorded in 5/15 patients and stabilization in 5/15 patients. In two patients progression of the disease was noted (one patient with progression of ocular pemphigoid, another patient with suspected automutilation). Premature withdrawal the drug was judged to be necessary in two patients with ocular surface disorders and in one patient with non-compliance., Conclusions: Topical Fk506 seems to be a promising new immunosuppressive drug for patients with atopic blepharokeratoconjunctivitis, Thygeson's superficial punctate keratitis and nummular adenoviral keratitis. Exact efficacy in these and other corneal and conjunctival inflammatory diseases has to be determined in randomised clinical studies. Before these studies may start the risk of side-effects must be reduced via an improvement of the drops.

Neoplastic transformation mediated by ras oncogenes is associated with deregulated expression of genes encoding, for example, various proteases, lysyl oxidase, and smooth-muscle alpha-actin. To define the role of these genes in the initiation or maintenance of the ras-transformed state, we compared their steady-state mRNA levels in two different sets of preneoplastic fibroblast lines, ras-transformed clones, and phenotypic revertants derived from them. Compared with the preneoplastic fibroblasts, the ras-transformed derivatives exhibited elevated levels of cathepsin L (major excreted protein), transin (stromelysin I, matrix metalloproteinase-3), and collagenase I (matrix metalloproteinase-1) mRNA but undetectable levels of lysyl oxidase mRNA. Partial restoration of lysyl oxidase transcription was observed in four of five phenotypic revertants derived from rat FE-8 and NIHpEJcl3 cells. The elevated levels of transin mRNA found in NIHpEJcl3 cells were diminished to the pretransformation level in interferon revertants but were not reduced in phenotypic rat FE-8 revertants expressing a high level of the ras oncoprotein. High steady-state levels of collagenase I mRNA were dependent on ras expression but were not closely associated with the transformed phenotype. High levels of cathepsin L mRNA were associated with neither high ras expression nor neoplastic transformation. The downregulation of smooth-muscle alpha-actin, characteristic of transformed cell lines, was not reversible in phenotypic revertants.

We analysed 39 prostatic carcinomas for loss of heterozygosity on chromosomal arms 8p, 10q, 16q, 17p and 18q and for mutations in the p53 anti-oncogene. Loss of heterozygosity (LOH) on 8p was detected in one out of 5 informative tumors, LOH on 16q in 3 out of 21 tumors, LOH on 17p in 2 out of 18 tumors, and LOH on 18q in 2 out of 17 tumors. No deletions were observed on 10q in 14 informative tumors. p53 alterations occurred in 3 out of 38 examined tumors, comprising two point mutations and a small deletion. Chromosomal deletions and p53 mutations were confined to locally invasive prostatic carcinomas, suggesting that they are associated with the progression of some prostate cancers rather than with tumor initiation.

We have analyzed the antiproliferative activity of the p53 tumor suppressor gene in human tumor cell lines harboring activated ras genes. The levels of p53 protein and incorporation of bromodeoxyuridine in transiently transfected cells were determined simultaneously by flow cytometry. The human HT1080 fibrosarcoma, EJ bladder carcinoma, and SW480 colon carcinoma cell lines were equally sensitive toward wild-type p53-mediated inhibition of DNA synthesis, independent of the state of the endogenous p53 protein. Overexpression of p53 genes mutated at amino acid codon 143 resulted in increased proliferation of SW480 cells, which have two mutated endogenous p53 alleles. To mimic the genetic constitution of an evolving tumor cell that has sustained a mutation in one p53 allele, we coexpressed both wild-type and mutant p53 genes controlled by strong viral promoters in HT1080 cells. Transiently transfected cells showed a reduced bromodeoxyuridine uptake similar to cells into which only wild-type p53 had been introduced. The wild-type p53 gene is a dominant growth suppressor over the mutant in all three different cell lines analyzed. By immunoprecipitation with antibodies PAb 122, PAb 420, and PAb 1620, we demonstrate the presence of both the mutant and wild-type conformations of the p53 protein in the transfected cells.

Expression of a transforming Ha-ras by dexamethasone in NIH3T3 cells transfected with a glucocorticoid-inducible Ha-ras construct results in a rapid desensitization of the intracellular Ca(2+)-mobilizing system to bombesin. This effect precedes the down-modulation of inositol trisphosphate (IP3) formation by several hours and is, therefore, not explained by an uncoupling of phosphoinositidase C. It is demonstrated that expression of Ha-ras attenuates the Ca(2+)-release by IP3 in permeabilized cells. The IP3 concentration required for half-maximal Ca(2+)-release is doubled in Ha-ras expressing cells. Maximal Ca(2+)-release which is obtained with 2 microM IP3 in control cells requires 10 microM IP3 in cells expressing Ha-ras. The desensitization of the IP3 receptors coincides with the desensitization of the Ca(2+)-mobilizing system to bombesin. The results indicate that the Ha-ras mediated desensitization of the Ca(2+)-releasing system to bombesin is--at least in part--caused by a decrease in the affinity of the IP3 receptor to inositol trisphosphate.

Hexadecylphosphocholine (HePC) inhibits protein kinase C (PKC) from NIH3T3 cells in cell-free extracts with a 50% inhibitory concentration of about 7 microM. Inhibition is competitive with regard to phosphatidylserine with a Ki of 0.59 microM. In order to determine whether HePC affects PKC in intact cells, the bombesin or tetradecanoylphorbolacetate-induced, PKC-mediated activation of the Na+/H(+)-antiporter was determined. It is demonstrated that HePC causes a drastic inhibition of this enzyme indicating a similar sensitivity of PKC to HePC in intact cells compared to cell-free extracts. In addition to the effects on PKC, treatment of NIH3T3 cells with HePC depresses the bombesin-induced formation of inositol 1,4,5-trisphosphate and the concomitant mobilization of intracellular Ca2+. Dose-response curves for the inhibition of inositol 1,4,5-trisphosphate formation and Ca2+ mobilization reveal 50% inhibitory concentrations of 2 or 5 microM, respectively. Polyphosphorylated phosphoinositides accumulate in HePC-treated cells indicating that the depression of inositol 1,4,5-trisphosphate generation is not caused by an inhibition of phosphoinositide kinases. Addition of bombesin to HePC-treated cells in the presence of LiCl revealed no evidence for an accelerated rate of inositol 1,4,5-trisphosphate turnover by the phospholipid analogue. It is concluded that HePC inhibits phosphoinositidase C in intact cells. The data strongly suggest that the growth-inhibitory effect of HePC is at least in part explained by the interference with mitogenic signal transduction.

Nitrogen mustard (N-mustard) inhibits the ouabain-sensitive and the furosemide-sensitive Rb uptake of Ehrlich ascites tumor cells, whereas the transport, which is resistant to both inhibitors, is not affected by the alkylating agent. At N-mustard concentrations below 10 microM, the reduction in Rb uptake is predominantly due to an interference with the furosemide-sensitive system. The dose response curve for the inhibition by N-mustard of the furosemide-sensitive Rb uptake closely parallels the dose response curve for the anti-tumor activity of the alkylating drug. This is in contrast to the behaviour of the ouabain-sensitive Rb transport. The inhibition of the furosemide-sensitive Rb uptake is expressed much less in cells which are resistant to N-mustard. The recovery of the furosemide-sensitive transport system after a single exposure to N-mustard is relatively slow and characterized by an initial 4 h lag period, whereas the repair of DNA-interstrand cross-links starts immediately after removal of the drug. At mM concentrations furosemide blocks the multiplication of Ehrlich ascites tumor cells. However, lower concentrations of furosemide which cause a 50% reduction in the furosemide-sensitive Rb uptake do not interfere with cell proliferation. This is in contrast to the behaviour of N-mustard which exerts a clear-cut depression of cell growth at concentrations leading to a 50% inhibition of the furosemide-sensitive Rb transport. It is concluded, therefore, that the inhibition of the furosemide-sensitive system alone is not sufficient to explain the anti-tumor activity of the alkylating agent. The effect is discussed as part of a more extended N-mustard-induced membrane alteration which may be important for the growth inhibitory effect of the alkylating agent.