Rama Malaviya

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

Histamine as an Autocrine Regulator of Leukemic Cell Proliferation

We examined leukemic lymphocyte precursors from ALL patients as well as immortalized ALL cell lines for cytoplasmic histamine expression. The histamine levels ranged from 10.8 pg/106 cells to 82.2 pg/106 cells in ALL cell lines (N=4) and from 12.5 pg/106 cells to 1235.4 pg/106 cells for primary leukemic cells from ALL patients (N=13). The presence of histamine in the cytoplasm of these ALL cells was also confirmed by immunostaining using a polyclonal rabbit anti-histamine antibody. Notably, the histamine receptor blocker diphenhydramine inhibited the clonogenic growth of ALL cells by <90% prompting the hypothesis that histamine may be an autocrine regulator of ALL cell proliferation. Our study suggests that histamine receptor blockers may therefore be useful for the treatment of therapy-refractory ALL.

Anti-Inflammatory Activity of 2,4,6-Trihydroxy-α-p-Methoxyphenyl- acetophenone (Compound D-58)

Background: Active oxygen radicals as well as a variety of cytosolic protein tyrosine kinases play a role in the regulation of prostaglandin E2 (PGE2), a key inflammatory mediator, released by skin cells in response to irradiation with ultraviolet B light (UVB). Identification of chemical compounds that can interrupt such events may provide a basis for the development of potent anti-inflammatory agents. Objective: To investigate the effect of a novel genistein analog, 2,4,6-trihydroxy-α-p-methoxyphenylacetophenone, with antioxidant property (compound D-58), on UVB-induced inflammatory responses. Methods: Epidermal cell cultures were irradiated with UVB both in the presence and absence of compound D-58 and the PGE2 released in the medium was determined by ELISA. For in vivo studies, skin inflammation was induced in mice either by carrageenan challenge of the air pouch or by an acute exposure of skin to UVB radiation. The resulting inflammatory mediator release, skin edema and the histological changes of the skin were determined both in the presence and absence of compound D-58. Results: Compound D-58 treatment effectively inhibited the development of edema and histological changes in the skin of UVB-irradiated mice as well as the release of PGE2 in vitro as well as in vivo. Conclusion: Compound D-58 (2,4,6-trihydroxy-α-p-methoxyphenylacetophenone) has potent anti-inflammatory properties.

The effect of receptor-mediated stimulation on the translocation of 5-lipoxygenase

It has been shown that calcium ionophore-mediated activation of the 5-lipoxygenase is followed by an extensive loss of enzyme activity. We investigated whether IgE/antigen stimulation of mast cells leads to a similar inactivation. This challenge caused a minor loss of 5-lipoxygenase activity as compared to A23187. Both stimuli resulted in the synthesis of similar amounts of leukotrienes. Immunoblot experiments showed that after antigen, the membrane association of the enzyme was reversible. In contrast, with A23187 translocation continued during the time of observation (60 min). Calcium chelators, added after A23187 stimulation, stopped the enzyme inactivation and reversed the membrane binding. The data suggest that the continuous, high intracellular calcium levels initiated by A23187 inactivate the enzyme, while the transient increase in calcium during receptor-mediated stimulation is sufficient to activate the 5-lipoxygenase but not to extensively inactivate it.