TLR9 re-expression in cancer cells extends the S-phase and stabilizes p16(INK4a) protein expression.

Toll-like receptor 9 (TLR9) recognizes bacterial, viral or cell damage-associated DNA, which initiates innate immune responses. We have previously shown that TLR9 expression is downregulated in several viral induced cancers including HPV16-induced cervical neoplasia. Findings supported that downregulation of TLR9 expression is involved in loss of anti-viral innate immunity allowing an efficient viral replication. Here we investigated the role of TLR9 in altering the growth of transformed epithelial cells. Re-introducing TLR9 under the control of an exogenous promoter in cervical or head and neck cancer patient-derived cells reduced cell proliferation, colony formation and prevented independent growth of cells under soft agar. Neither TLR3, 7, nor the TLR adapter protein MyD88 expression had any effect on cell proliferation, indicating that TLR9 has a unique role in controlling cell growth. The reduction of cell growth was not due to apoptosis or necrosis, yet we observed that cells expressing TLR9 were slower in entering the S-phase of the cell cycle. Microarray-based gene expression profiling analysis highlighted a strong interferon (IFN) signature in TLR9-expressing head and neck cancer cells, with an increase in IFN-type I and IL-29 expression (IFN-type III), yet neither IFN-type I nor IL-29 production was responsible for the block in cell growth. We observed that the protein half-life of p16(INK4a) was increased in TLR9-expressing cells. Taken together, these data show for the first time that TLR9 affects the cell cycle by regulating p16(INK4a) post-translational modifications and highlights the role of TLR9 in the events that lead to carcinogenesis.

Effect of immunization against melatonin on prolactin concentrations and the timing of reproductive transitions in ewes.

The objective of this experiment was to develop a procedure for immunizing ewes against melatonin that would alter the effects of changing photoperiod on seasonal reproduction and prolactin secretion. Ewes were immunized against human serum albumin (HSA) as controls (n = 9) or a melatonin-human serum albumin conjugate (0.25 mg; n = 10) on December 14th (Day 0) and boosted 9 times. They were maintained on natural photoperiod and then transferred indoors and exposed to long days for 35 d, followed by short days for 146 d, long days for 93 d, and short days for a further 123 d. Antibody titers to melatonin (at a serum dilution of 1:1,250) were significantly higher in immunized ewes (27.3 +/- 6.6%) than controls (0.7 +/- 0.1%; P < 0.001). At the end of the experiment, antibody titers in immunized ewes (at dilution of 1:50) were higher in blood (43.7 +/- 8.2%) than in cerebrospinal fluid (10.8 +/- 3.9%; P < 0.05), and highly correlated (r2 = 0.746). Onset of the breeding season was advanced slightly after the second transfer from long to short days in immunized ewes (April 12 +/- 3 d) compared with controls (April 25 +/- 3 d; P < 0.05). Mean serum prolactin concentrations were lower (P < 0.05) in melatonin-immunized ewes compared with controls on natural photoperiod, after transfer from long to short days, during long days, and after the second transfer from long to short days. In conclusion, despite melatonin-immunization increasing antibody titers in blood and cerebrospinal fluid, and decreasing prolactin concentrations over much of the experiment, minimal effects on the timing of reproductive transitions in the ewes were evident. This discrepancy between the response of the prolactin and reproductive axes to melatonin immunization supports the hypothesis of a dual site of action of melatonin, with melatonin acting in the pituitary gland to mediate the effects of photoperiod on prolactin secretion and in the mediobasal hypothalamus to affect reproductive responses.

Active immunization against melatonin in Ile-de-France ewes and photoperiodic control of prolactin secretion and ovulatory activity.

An experiment was conducted to determine whether active immunization against melatonin could modify the perception of abrupt photoperiodic changes in ewes. Two groups each containing six intact Ile-de-France ewes were submitted to alternate periods of short days for 2.5 months and long days for 2.5 days for about 70 weeks. Three series of active immunizations against a melatonin conjugate were carried out during the first of the three long-day periods. Control ewes were actively immunized at the same time against human serum albumin. Blood samples were taken once a week throughout the experiment to measure antibody titre and affinity, and prolactin and progesterone concentrations. Sera of all treated ewes demonstrated higher antibody titres than those of control ewes. Antisera were highly specific, as evidenced by the absence of displacement of iodinated melatonin in the presence of ten melatonin metabolites. Significant effects of photoperiod and of the interaction between treatment and photoperiod on prolactin concentration were detected. Prolactin concentrations in plasma of the control ewes were high during long days and low during short days. However, in the treated ewes, with the exception of the first period of long days, prolactin concentrations were not influenced by photoperiodic changes. Ovulatory activity of control ewes, as demonstrated by progesterone measurements, was stimulated by short days and inhibited by long days. In contrast, ovulatory activity of treated ewes, after a response identical to that of control ewes after the first photoperiodic shift from long to short days, showed a complete desynchronization of ovulatory activity relative to photoperiodic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of melatonin receptors in the ram pars tuberalis: influence of light.

The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20 degrees C, but unstable at 28 or 37 degrees C; 3. melatonin and [127I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 10(10) l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25 +/- 0.30 vs 1.01 +/- 0.17 fmol/mm2 pars tuberalis, p less than 0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.