No abstract

Non-steroidal antioestrogens, such as tamoxifen, inhibit the growth of human breast cancer cells. The experiments described here compare and contrast the efficacy of tamoxifen and the 'pure' antioestrogen, ICI 164384, on the inhibition of proliferation of MCF-7 cells. Previous studies have shown that ICI 164384 has a greater maximal inhibitory effect than conventional antioestrogens on the growth of MCF-7 cells. Both types of compound block progression of cells through the cell cycle in the early G1 phase. These studies have been extended to measure the population distribution of antioestrogen-treated cells by the use of two-parameter flow cytometry. ICI 164384 proved to be more effective than tamoxifen in decreasing the proportion of actively growing cells in an asynchronous population. In cells grown in the complete absence of exogenous oestrogens, growth was stimulated by oestradiol, insulin, insulin-like growth factor-I (IGF-I) or transforming growth factor-alpha (TGF-alpha). The potent metabolite of tamoxifen, trans 4'-hydroxytamoxifen (4'-OHT), alone also stimulated growth, whereas ICI 164384 did not. Oestradiol and insulin added together demonstrated a clear synergistic enhancement of cell growth. Correspondingly, the stimulatory effect of 4'-OHT on growth was magnified in the presence of insulin, and a combination of ICI 164384 with insulin revealed a much weaker stimulatory action of the 'pure' antagonist. For both compounds the interaction with insulin was complex and characterized by a bell-shaped dose-response curve. However, for 4'-OHT at all concentrations in the range 1 pM-1 microM in the presence of insulin, cell numbers were greater than in cultures exposed to insulin alone. This was not the case for ICI 164384 which suggested that differences in efficacy may be due to interactions between oestrogen and growth factor-mediated mechanisms. Furthermore, ICI 164384 was more effective in inhibiting the action of IGF-I and TGF-alpha alone or in combination, although both antioestrogens produced a partial blockade of growth factor responses in the complete absence of oestradiol. It is concluded that the difference in efficacy between partial agonist and 'pure' antagonist antioestrogens to inhibit growth in vitro is consistent with the difference in the pharmacological profile of these compounds. The absence of stimulatory activity of ICI 164384 is of particular significance in reducing to a minimum the synergistic interaction between oestrogens and insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Pathogenesis-related protein-10 (PR10) is a ubiquitous small plant protein induced by microbial pathogens and abiotic stress that adversely contributes to the allergenic potency of many fruits and vegetables, including carrot. In this plant, two highly similar genes encoding PR10 isoforms have been isolated and designated as allergen Dau c 1.01 and Dau c 1.02. The aim of the study was to generate PR10-reduced hypoallergenic carrots by silencing either one of these genes in transgenic carrots by means of RNA interference (RNAi). The efficiency of gene silencing by stably expressed hairpin RNA (hnRNA) was documented by means of quantitative RT-PCR (qPCR) and immunoblotting. Quantification of the residual protein revealed that PR10 accumulation was strongly decreased compared with untransformed controls. Treatment of carrot plants with the PR protein-inducing chemical salicylic acid resulted in an increase of PR10 isoforms only in wild-type but not in Dau c 1-silenced mutants. The decrease of the allergenic potential in Dau c 1-silenced plants was sufficient to cause a reduced allergenic reactivity in patients with carrot allergy, as determined with skin prick tests (SPT). However, simultaneous silencing of multiple allergens will be required to design hypoallergenic carrots for the market. Our findings demonstrate the feasibility of creating low-allergenic food by using RNAi. This constitutes a reasonable approach to allergen avoidance.

Dau c 1 isoforms display distinct IgE epitope heterogeneity. Dau c 1.03 appears to contribute to the allergenicity of carrots and the manifestation of carrot allergy. The epitope diversity of different Dau c 1 isoforms should be considered for component-resolved diagnosis and gene silencing of carrot allergens.