The tumorigenic phenotype of a mutated form of Fc gamma RIIB1, lacking the ability to generate soluble receptor and allowing a low-level of ligand binding.

Non immunohematopoietic murine tumor cells ectopically expressing Fc gamma RIIB1 (B1) were recently shown to express a higher tumorigenicity phenotype than cells not expressing this receptor. Utilizing a genetic approach we studied the possible contribution of a soluble form of B1 to tumor enhancement. A mutated form of the B1, lacking the cleavage site responsible for the generation of soluble B1 was produced using gene splicing by overlap extension PCR. A deletion confirmed by sequence analysis from 172 to 178 residues was generated. Stable transfectants expressed the B1 deleted form (B1 Delta) both as specific RNA and as a membrane protein receptor allowing a low level of ligand binding. The soluble form of B1 was undetectable in tissue culture supernatants of Bib transfected cells while it was present in supernatants of wild type B1-transfectants. Stable B1 Delta transfectants were significantly more tumorigenic than negative control transfectants. Tumor incidence was almost as high as that of intact B1 and lagged in the latency period before the appearance of palpable tumors. It is suggested that the soluble B1 has a minimal contribution to tumor enhancement.

Assessment of stimulated and spontaneous adrenocorticotropin secretory dynamics identifies distinct components of cortisol feedback inhibition in healthy humans.

Corticosteroids inhibit ACTH secretion through diverse cellular mechanisms, including direct pituitary and indirect suprapituitary effects. Although exogenous CRH provides a useful assessment of corticotroph function, the suprapituitary component of ACTH regulation has been difficult to assess in humans. Naloxone (NAL) has been reported to stimulate ACTH secretion indirectly through the release of endogenous hypothalamic CRH, suggesting its potential application in the examination of suprapituitary regulation of ACTH secretory dynamics. We sought to examine the inhibitory effects of corticosteroids on kinetic parameters of ACTH secretion, assessed by a deconvolution method, in healthy human subjects. We also sought to directly compare the ACTH responses to serial administration of human CRH and NAL as well as spontaneously occurring ACTH pulses to distinguish pituitary and suprapituitary components of hypothalamic-pituitary-adrenal regulation. Normal healthy subjects (n = 11) received hCRH (0.4 microgram/kg) at 1800 h and then NAL (65 micrograms/kg) at 1930 h, respectively, on 3 separate study days: placebo pretreatment plus CRH/NAL stimulation, metyrapone (MET) pretreatment plus CRH/NAL, or MET alone. Plasma ACTH and serum cortisol were assessed at frequent (every 10 min) intervals during CRH/NAL or placebo infusions (1800-2100 h) on all 3 study days, and deconvolution analysis was performed to determine kinetic parameters of endogenous and stimulated ACTH secretion. Suppression of endogenous cortisol secretion with MET significantly increased both continuous (basal secretion rate) and pulsatile CRH- and NAL-stimulated ACTH bursts (P < 0.05). The increase in total ACTH secreted per burst was related to two distinct effects of cortisol regulating the amplitude (maximum secretion rate) and half-duration (P < 0.05) of secretory bursts. The ACTH responses to CRH and NAL for individual subjects were significantly and positively correlated in both placebo pretreatment plus CRH/NAL stimulation and MET pretreatment plus CRH/NAL studies (P < 0.01). MET administration disproportionately increased the ACTH response to NAL, producing a significant increase (P < 0.01) in the slope of the regression relating ACTH responses to CRH and NAL. The following conclusions were made: 1) endogenous cortisol secretion, even at levels associated with relatively low serum cortisol concentrations, exerts a significant negative feedback effect on both continuous and pulsatile ACTH secretion; 2) cortisol inhibits pulsatile ACTH secretion through distinct regulatory mechanisms that independently modulate both the mass and the duration of ACTH secretory bursts; 3) the differential sensitivity of the CRH- and NAL-stimulated ACTH responses to MET administration suggests that both pituitary and suprapituitary components of the hypothalmic-pituitary-adrenal axis are sensitive to negative regulation over a rapid or intermediate temporal domain. Endogenous cortisol modulates multiple components of dynamical ACTH secretion through composite effects that appear to be mediated through structurally and functionally distinct regulatory domains.

Contribution of the intracellular domain of murine Fc-gamma receptor type IIB1 to its tumor-enhancing potential.

We have previously shown that Fc gamma receptor type II B1 (Fc(gamma)RIIB1), when expressed on non-lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of Fc(gamma)RIIB1 in the enhancement of the malignant phenotype of polyoma-transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: Fc(gamma)RIIB1, a full-length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; Fc(gamma)RIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and Fc(gamma)RIIB1-CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand-bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble Fc(gamma)R, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1-expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than Fc(gamma)R-negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1-specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

The murine Fc-gamma (Fc gamma) receptor type II B1 is a tumorigenicity-enhancing factor in polyoma-virus-transformed 3T3 cells.

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fc gamma receptor type II B I (Fc gamma RIIB I) functions as a progression-enhancing factor when expressed ectopically on non-lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express Fc gamma RII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific Fc gamma RII transcript present in tumor cells was identified in this report as Fc gamma RIIB I (BI). In order to determine whether or not the ectopically expressed Fc gamma RII plays a role in the progression of these transformed cells, PyV-transformed 3T3 cells were transfected with BI-cDNA. The BI transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co-transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. Fc gamma RIIB I expressors exhibited a significantly higher tumorigenic phenotype than FcR-negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of Fc gamma RIIB I to act as a potentially tumorgenicity-enhancing factor was also demonstrated as Fc gamma RII was expressed by tumor cells, originating from inoculated Fc gamma RIIB I-transfected cells, or from inoculation of a mixture of receptor-positive and -negative cells. B I-expressing cells dominated the tumor-cell population over non-expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Elevated rectal temperature produced by all-night bright light is reversed by melatonin infusion in men.

Early morning rectal body temperature is lowest when melatonin levels are highest in humans. Although pharmacological doses of melatonin are hypothermic in humans, the relationship between endogenous melatonin and temperature level has not been investigated. We measured rectal body temperature in nine normal men whose melatonin levels were suppressed by all-night sleep deprivation in bright light and compared values with those seen in sleep in the dark, sleep deprivation in dim light (to control for the stimulatory effect of wakefulness on temperature), and sleep deprivation in bright light with an infusion of exogenous melatonin that replicated endogenous levels. Minimum rectal temperature, calculated from smoothed temperature data from 2300 to 0515 h, was greater in bright-light sleep deprivation, resulting in suppression of melatonin, than in conditions of sleep deprivation in dim light or sleep in the dark. An exogenous melatonin infusion in bright light returned the minimum temperature to that seen in dim-light sleep deprivation. A nonsignificant elevation in mean and minimum temperature was noted in all conditions of sleep deprivation relative to sleep. We conclude that melatonin secretion contributes to the lowering of core body temperature seen in the early morning in humans.

Sleep deprivation reduces LH secretion in men independently of melatonin.

Melatonin affects gonadal function in non-primate mammals. Confirmatory data in man are not available. We assessed melatonin's acute effects on luteinizing hormone secretion in 17 normal men. We studied these men in conditions of sleep in the dark, and sleep deprivation in bright light, dim light, and bright light combined with a physiologically relevant infusion of melatonin, while measuring blood levels of immunoreactive LH every 20 min for 7 h. We compared overnight LH secretion, and LH pulse frequency, amplitude, length, interval and area under the curve using a modification of the PULSAR peak identification program, among the four treatments. Areas under the curve for peaks in all three conditions of sleep deprivation were lower than in normal sleep. The presence or absence of melatonin had no additional effect. We conclude that acute suppression of melatonin does not affect LH pulse parameters in normal man, but that sleep deprivation may reduce the amount of LH secreted per pulse.

Lack of an acute modulatory effect of melatonin on human nocturnal thyrotropin and cortisol secretion.

Using a recently developed model for investigating the neuroendocrine role of melatonin in man, we studied melatonin's effect on the nocturnal secretion of thyrotropin and cortisol in 17 normal male volunteers. The model consists of sleep in the dark and all-night sleep deprivation in conditions of: bright light with and without a melatonin infusion, and dim light. We have improved our infusion paradigm so that levels of melatonin during infusion are now indistinguishable from those occurring during sleep in the dark or dim light sleep deprivation. Sleep deprivation per se raised TSH levels compared to normal sleep. However, the three conditions of sleep deprivation could not be distinguished from each other, which suggests that the suppression of TSH by sleep (or the stimulation of TSH by sleep deprivation) is not mediated by melatonin. Cortisol secretion was unaffected by sleep deprivation regardless of melatonin's presence or absence. However, a difference in the pattern of secretion of cortisol in the sleep condition in the early morning (compared to the sleep deprivation conditions) was noted. These data do not implicate melatonin in the acute regulation of TSH or cortisol in normal man. These data also provide a method of melatonin infusion that replicates the pattern and levels seen in sleep.

A model for the study of the acute effects of melatonin in man.

The role of the pineal hormone melatonin in human physiology is uncertain. Previous studies correlated plasma melatonin levels with several physiological parameters or determined the responses to pharmacological doses of melatonin during daylight hours. We established an acute model that is more rigorously physiological. Constant nocturnal bright light in sleep-deprived normal men resulted in low plasma melatonin levels throughout the night, in contrast to sleep in the dark and dim light sleep deprivation nights. Subsequently, melatonin was infused during bright light exposure to approximate physiological levels. Plasma GH and PRL measurements in these four conditions revealed an effect of sleep deprivation independent of the presence or absence of melatonin. A subsample of these men had an intermediate level of melatonin suppression with 500 lux light intensity, relative to those during sleep and bright light. The results suggest that melatonin has no acute modulatory effect on the secretion of these two sleep-related hormones.