A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death.

We have discovered that the accumulation of an anti-calcitonin receptor (anti-CTR) antibody conjugated to a fluorophore (mAb2C4:AF568) provides a robust signal for cells undergoing apoptotic programmed cell death (PCD). PCD is an absolute requirement for normal development of metazoan organisms. PCD is a hallmark of common diseases such as cardiovascular disease and tissue rejection in graft versus host pathologies, and chemotherapeutics work by increasing PCD. This robust signal or high fluorescent events were verified by confocal microscopy and flow cytometry in several cell lines and a primary culture in which PCD had been induced. In Jurkat cells, GBM-L2 and MG63 cells, the percentage undergoing PCD that were positive for both mAb2C4:AF568 and annexin V ranged between 70 and >90%. In MG63 cells induced for the preapoptotic cell stress response (PACSR), the normal expression of α-tubulin, a key structural component of the cytoskeleton, and accumulation of mAb2C4:AF568 were mutually exclusive. Our data support a model in which CTR is upregulated during PACSR and recycles to the plasma membrane with apoptosis. In cells committed to apoptosis (α-tubulin negative), there is accumulation of the CTR-ligand mAb2C4:AF568 generating a high fluorescent event. The reagent mAb2C4:AF568 effectively identifies a novel event linked to apoptosis.

Impaired spatial learning in aged rats is associated with loss of p75-positive neurons in the basal forebrain.

We investigated age-related changes in the number and size of neurons positive for the p75 neurotrophin receptor in the cholinergic basal forebrain of female Dark Agouti rats. Since the integrity of these neurons is known to be closely associated with performance in tests of spatial learning ability, we also investigated the incidence of age-related spatial learning impairments, using the Barnes maze. Spatial learning impairments occurred with increasing frequency with age. No rats showed impairment at six months, but 50% were impaired at 14 months and 71% at 26 months. There was no correlation between age and decreased number of p75-positive neurons in the rostral basal forebrain, which consists of the medial septum and vertical limb of the diagonal band of Broca. In the caudal basal forebrain, which consists of the horizontal limb and the nucleus of Meynert, there was a 13% reduction in the number of p75-positive neurons at 17 months compared to six months, and a 30% reduction at 26 months. There was a strong correlation between the presence of spatial learning impairment and a reduction in the number of p75-positive neurons. This correlation was most evident in the rostral basal forebrain, but was also present in the caudal basal forebrain. In the rostral basal forebrain, all learning impaired rats had fewer p75-positive neurons than the average number in unimpaired rats. A close correspondence between the presence of p75 and choline acetyltransferase was evident in basal forebrain neurons of learning-impaired and unimpaired rats. Gross pathological changes to the morphology of p75-positive neurons were relatively frequent in learning-impaired rats. These changes consisted of hypertrophy, appearance of vacuoles, and marginalisation of the cytoplasm. The results indicate the susceptibility of p75-positive neurons to degenerative changes with aging, and show that the loss of these neurons in the basal forebrain was strongly correlated with impairment in spatial learning.

Enlarged cholinergic forebrain neurons and improved spatial learning in p75 knockout mice.

The p75 low affinity neurotrophin receptor (p75) can induce apoptosis in various neuronal and glial cell types. Because p75 is expressed in the cholinergic neurons of the basal forebrain, p75 knockout mice may be expected to show an increased number of neurons in this region. Previous studies, however, have produced conflicting results, suggesting that genetic background and choice of control mice are critical. To try to clarify the conflicting results from previous reports, we undertook a further study of the basal forebrain in p75 knockout mice, paying particular attention to the use of genetically valid controls. The genetic backgrounds of p75 knockout and control mice used in this study were identical at 95% of loci. There was a small decrease in the number of cholinergic basal forebrain neurons in p75 knockout mice at four months of age compared with controls. This difference was no longer apparent at 15 months due to a reduction in numbers in control mice between the ages of 4 and 15 months. Cholinergic cell size in the basal forebrain was markedly increased in p75 knockout mice compared with controls. Spatial learning performance was consistently better in p75 knockout mice than in controls, and did not show any deterioration with age. The results indicate that p75 exerts a negative influence on the size of cholinergic forebrain neurons, but little effect on neuronal numbers. The markedly better spatial learning suggests that the function, as well as the size, of cholinergic neurons is negatively modulated by p75.

Cytogenetic effects of occupational exposure in the peripheral blood lymphocytes of pesticide sprayers.

The potential clastogenic effect of pesticides was investigated in 56 (29 indoor and 27 outdoor) agricultural workers exposed to complex chemical mixtures. Exposed and referent subjects were selected from the same geographical area located in Ionia, province of Thessaloniki, Greece. Chromosome aberrations (CA) and sister chromatid exchanges (SCE), were studied in peripheral lymphocytes. Comparison between workers and control group revealed that the individuals exposed to pesticides showed substantial clastogenic effects (CA = 2.66% compared to 0.53%, P < 0.001), in their lymphocytes without indication of increases in their basal frequency of SCE. Moreover, the condition of exposure has been found to influence the CA frequency. It was observed that individuals working exclusively in greenhouses (confined spaces) showed higher CA levels than subjects working in open fields (3.37 versus 1.88, P < 0.01). No significant difference in their expression of CA between smokers and non-smokers was found. The present chromosome study included workers living in the close vicinity of a large industrial zone near Thessaloniki. The percentage of CA in these indoor sprayers was higher compared to our previous study carried out in a different area of Thessaloniki, free of industrial plants (3.37% compared to 2.14%, P < 0.02).

Cytogenetic damage by melphalan and hyperthermia in patients with an initial epileptic attack.

Sister-chromatid exchanges (SCEs) and cell kinetics in cultured lymphocytes of patients with an initial epileptic attack, and prior to any anticonvulsant treatment, were studied. Spontaneous melphalan (MEL) and MEL-hyperthermia (MEL-HYP) induced SCE frequencies have been studied in 18 adults with an initial epileptic seizure. Fifteen age and sex matched healthy subjects were used as the control group. The incidence of spontaneous SCEs in lymphocytes from epileptics was not significantly greater than in those from the control subjects. However, when exposed to MEL in vitro, cells from both groups showed an increase in SCE frequency. When exposed to MEL and HYP (41 degrees C for 3 h) in vitro, cells from both groups showed a further increase in SCE frequency with yields from epileptics higher (P less than 0.05) than from controls. HYP in combination with MEL enhanced synergistically SCEs and cell division delays in both groups with synergistic effects in cells from epileptics (P less than 0.01 and P less than 0.01 respectively) higher than from controls (P less than 0.05 and P less than 0.05 respectively.

Frequencies of chromosomal aberrations in pesticide sprayers working in plastic green houses.

The frequency of chromosome aberrations was studied in peripheral blood lymphocytes of 29 workers occupationally exposed to a mixture of pesticides and in 14 age- and sex-matched healthy controls. There was a significant increase in chromosome aberrations in sprayers when compared to unexposed persons (2.39% compared to 0.54%). No positive correlation between the frequency of chromosome aberrations and the duration of exposure was observed. No significant difference between smokers and non-smokers was found.

Enhanced genotoxicity by melphalan and hyperthermia in chronic heroin addicts.

Spontaneous melphalan- (MEL-) and MEL-hyperthermia- (MEL-HYP-) induced sister chromatid exchange (SCE) frequencies have been studied in 12 chronic heroin addicts (HER AD) and in 12 age- and sex-matched healthy controls. The incidence of spontaneous SCEs in lymphocytes from the HER AD was significantly greater (P less than 0.001) than those from the control subjects. When exposed to MEL in vitro, cells from both groups showed an increase in SCE frequency with yields from HER AD higher than those from controls. When exposed to MEL and hyperthermia (41.5 degrees C for 3 h) in vitro, cells from both groups showed further increases in SCE frequency with yields from HER AD higher than those from controls. We observed that hyperthermia (HYP) in combination with MEL synergistically enhances SCEs and cell division delays in both groups, with the synergistic effects in cells from HER AD higher than those from controls.

Enhancement of cytogenetic damage by inhibitors of poly(ADP-ribose)polymerase in human lymphocytes exposed to antineoplastics in vivo and in vitro.

The effect of benzamide (B) and 3-aminobenzamide (3-AB) on sister chromatid exchanges (SCEs) and cell kinetics induced in vitro by melphalan (MELPH) or thiotepa (THIO) was studied in normal human lymphocytes. The combined treatments with either MELPH or THIO plus B or 3-AB showed the potentiating ability on SCE rates and the ability to induce cell division delays of the latter chemicals. In a combined in vivo and in vitro study, lymphocytes taken from six cancer patients who had been given cytoxan by injection 2 hr before and then treated with theophylline (THEOPH) or B or 3-AB in vitro were found to have synergistically increased exchange rates and cell division delays. The frequency of SCEs in the patients own lymphocytes with and without exposure to inhibitor of Poly(ADP-ribose) polymerase (P(ADPR)polymerase) was determined before the cytostatic therapy and was used as a control for later comparison in each individual case. These results further substantiate the use of this approach for detecting the induction of cytogenetic damage concerning controlled mutagen human exposure in combined in vivo and in vitro studies. Chemically induced cytotoxicity manifested as an alteration (division delay) in cell kinetics and as synergistic DNA damage by cytostatics and inhibitors of P(ADPR)polymerase may be of use in the treatment of human cancer.

Synergistic induction of sister-chromatid exchanges in lymphocytes from normal subjects and from patients under cytostatic therapy by inhibitors of poly(ADP-ribose)polymerase and antitumour agents.

The effects of nicotinamide on SCE rates induced in vitro by chlorambucil (CBC or melphalan (MELPH) or mitomycin C (MMC) was studied. The combined treatments with either CBC or MELPH or MMC and nicotinamide showed the potentiating ability of the latter drug. Theophylline and MELPH were also found to act synergistically on the induction of SCEs. In a combined in vivo and in vitro study, lymphocytes taken from 7 cancer patients who had been given cytoxan by injection 3 h before, were treated with nicotinamide or diphylline (DP) in vitro, and found to have synergistically increased exchange rates. This has implications for interpreting the repair processes involved, for monitoring drug combinations that synergistically damage DNA in vivo and in vitro and for identifying interindividual variation in the response to the treatment.

Practical applications of the SCE studies for guiding and improving chemotherapy.

The effect of diphylline (DP) or (1,2-dihydroxy-3-propyl)-theophylline and theobromine (TB) on sister chromatid exchange (SCE) rates induced in vitro by cytosine arabinoside (AraC) was studied in normal human lymphocytes. The combined treatments with AraC plus DP or TB showed the potentiating ability of the latter drugs. In a combined in vivo and in vitro study, lymphocytes taken from 14 patients suffering from various types of cancer who had been given Cytoxan (5 patients) or AraC (9 patients) by injection 3 hr before and then treated with DP or TB in vitro were found to have synergistically increased exchange rates. This has implications for interpreting the repair processes involved and for monitoring drug combinations that synergistically damage DNA in vivo and in vitro.