Phospholipase D (PLD) drives cell invasion, tumor growth and metastasis in a human breast cancer xenograph model.

Breast cancer is one of the most common malignancies in human females in the world. One protein that has elevated enzymatic lipase activity in breast cancers in vitro is phospholipase D (PLD), which is also involved in cell migration. We demonstrate that the PLD2 isoform, which was analyzed directly in the tumors, is crucial for cell invasion that contributes critically to the growth and development of breast tumors and lung metastases in vivo. We used three complementary strategies in a SCID mouse model and also addressed the underlying molecular mechanism. First, the PLD2 gene was silenced in highly metastatic, aggressive breast cancer cells (MDA-MB-231) with lentivirus-based short hairpin RNA, which were xenotransplanted in SCID mice. The resulting mouse primary mammary tumors were reduced in size (65%, P<0.05) and their onset delayed when compared with control tumors. Second, we stably overexpressed PLD2 in low-invasive breast cancer cells (MCF-7) with a biscistronic MIEG retroviral vector and observed that these cells were converted into a highly aggressive phenotype, as primary tumors that formed following xenotransplantation were larger, grew faster and developed lung metastases more readily. Third, we implanted osmotic pumps into SCID xenotransplanted mice that delivered two different small-molecule inhibitors of PLD activity (5-fluoro-2-indolyl des-chlorohalopemide and N-[2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]dec-8-yl)ethyl]-2-naphthalenecarboxamide). These inhibitors led to significant (>70%, P<0.05) inhibition of primary tumor growth, metastatic axillary tumors and lung metastases. In order to define the underlying mechanism, we determined that the machinery of PLD-induced cell invasion is mediated by phosphatidic acid, Wiscott-Aldrich Syndrome protein, growth receptor-bound protein 2 and Rac2 signaling events that ultimately affect actin polymerization and cell invasion. In summary, this study shows for the first time that PLD2 has a central role in the development, metastasis and level of aggressiveness of breast cancer, raising the possibility that PLD2 could be used as a new therapeutic target.

Contribution of raloxifene and calcium and vitamin D3 supplementation to the increase of the degree of mineralization of bone in postmenopausal women.

Raloxifene has been shown to increase bone mineral density and reduce the risk of vertebral fracture in postmenopausal women with osteoporosis. In this study, we report the results of the first prospective longitudinal study to evaluate the mean degree of mineralization of bone (MDMB) in a group of patients enrolled in the Multiple Outcomes of Raloxifene Evaluation trial. Patients were randomly assigned to one of three treatment groups: placebo (n = 24), raloxifene 60 mg/d (RLX60; n = 22), or raloxifene 120 mg/d (RLX120; n = 18). All patients received daily calcium (500 mg) and vitamin D(3) (400-600 IU) supplementation for the duration of the study. Iliac crest biopsies were taken at baseline and after 2 yr of treatment. Quantitative microradiography was used to analyze the biopsy specimens and revealed a statistically significant (P < 0.05) mean percentage increase in total MDMB of 7.0, 5.3, and 5% for RLX60-, RLX120-, and placebo-treated patients, respectively, compared with baseline. Raloxifene treatment was found to shift the distribution of total bone mineral to higher values of MDMB (RLX60, 29%; RLX120, 8%) with greater heterogeneity, compared with placebo. The profile of MDMB observed in biopsies after treatment with placebo and raloxifene, compared with baseline, closely resembles physiological premenopausal bone.

Characterization of the DNA polymerase and thymidine kinase genesof herpes simplex virus isolates from AIDS patients in whom acyclovirand foscarnet therapy sequentially failed.

Herpes simplex virus (HSV) isolates were characterized from 8 AIDS patients in whom acyclovir and foscarnet therapy sequentially failed. The 6 postacyclovir (prefoscarnet) HSV isolates were resistant to acyclovir and susceptible to foscarnet. Of the 9 postfoscarnet isolates, 8 were foscarnet-resistant and acyclovir-susceptible, 1 was resistant to both drugs. Acyclovir- or foscarnet-resistant isolates retained susceptibility to cidofovir. The acyclovir-resistant isolates contained single-base substitutions or frameshift mutations in G or C homopolymer nucleotide repeats of the thymidine kinase gene. In contrast, the foscarnet-resistant strains contained single-base substitutions in conserved (II, III, or VI) or, more rarely, nonconserved (between I and VII) regions of the DNA polymerase (pol) gene. The single isolate exhibiting resistance to acyclovir and foscarnet contained mutations in both genes. In this study of clinical HSV isolates, DNA pol mutations conferring foscarnet resistance were not associated with decreased acyclovir or cidofovir susceptibility.

Chemical and X-ray analysis of fluoride, phosphorus, and calcium in human foetal blood and hard tissues.

To evaluate the beneficial effect of prenatal fluoride supplementation, the presence of fluoride in hard tissues in two populations of human foetuses coming from fluoridated (> or = 0.7 parts/10(6) F in drinking water) and non-fluoridated areas (< or = 0.1 parts/10(6) F in drinking water) were compared by chemical analysis and X-ray microanalysis. The fluoride concentrations measured in maternal and venous cord blood confirmed that placental transfer of fluoride was passive when fluoride intake was low. Total fluoride contents of tooth germs and mandibular bone appeared to increase with fluoride level in drinking water. However, these concentrations were too low to be detected by X-ray microanalysis. Phosphorus and calcium total contents were identical in mandibular and femoral bone of both populations. In incisor germs, phosphorus and calcium concentrations in enamel and dentine close to the amelodentinal junction did not differ significantly between the two populations. It is suggested that the low fluoride concentrations in enamel and dentine formed in utero would not have a significant effect on acid solubility.

Strontium distribution and interactions with bone mineral in monkey iliac bone after strontium salt (S 12911) administration.

The analysis of the interaction of strontium (Sr) with bone mineral is of interest because a new agent containing Sr (S 12911) has shown positive effects on bone mass in various animal models of osteoporosis and is currently being developed for preventive and curative treatment of postmenopausal osteoporosis. Iliac bone samples were obtained from 20 male monkeys: 4 untreated control animals, 12 animals sacrificed at the end of a 13-week treatment with high dose levels of S 12911 (750, 275, or 100 mg/kg/day orally), and 4 animals sacrificed 6 weeks after the end of a 13-week treatment with S 12911 (750 or 100 mg/kg/day orally). The distribution of Sr was determined and quantified by X-ray microanalysis. Changes at the crystal level were evaluated by X-ray diffraction and Raman microspectrometry. In the control animals, traces of Sr were found to be homogeneously distributed throughout the bone tissue. In the treated monkeys, Sr could only be detected in calcified matrix. In monkeys sacrificed at the end of the treatment, Sr was found to be dose-dependently incorporated into the mineral substance of the compact and cancellous bone. Sr was heterogeneously distributed with three to four times more Sr in new than in old compact bone, and approximately two and a half times more Sr in new than in old cancellous bone. The bone Sr content dramatically decreased in the animals sacrificed 6 weeks after the end of the treatment. Diffraction showed no significant changes in the characteristics of the crystal lattice. Sr appeared to be easily exchangeable from bone mineral and was slightly linked to mature crystals through ionic substitutions. Even at the highest dose level tested, less than 1 calcium ion out of 10 was substituted by 1 Sr ion in each crystal. In conclusion, taken up by bone, Sr was heterogeneously distributed with a higher concentration in new than in old bone but induced no major modifications of the bone mineral (crystallinity, crystal structure) at the crystal level. As a result, a treatment with S 12911 Sr salt should not induce any alteration of bone mineral.

Dose effects on ewe bone remodeling of short-term sodium fluoride administration--a histomorphometric and biochemical study.

The early effects of two doses of sodium fluoride (NaF) on bone remodeling was studied in 14 ewes divided into two groups. Group I received orally 1 mg NaF/kg/day and group II received a five-fold greater dose. No calcium supplement was given. Transiliac bone biopsies and blood samples were taken before treatment (T0) and after 45 (T45) days of treatment. Bone fluoride content significantly increased in group II. In both groups, a significant decrease of serum calcium and phosphorus, and a slight but nonsignificant augmentation in serum parathyroid hormone were noted. Osteoid perimeter and area were significantly increased. The osteoid width significantly increased in both groups, but was twice higher in group II than I. At T45, the osteoblast perimeter increased in both groups. Osteoid perimeter was significantly correlated with serum osteocalcin values (r = 0.74; p less than 0.001) and bone fluoride content (r = 0.64; p less than 0.01). The bone formation rate at tissue level tended to increase in both groups. Concerning the apposition rate, a decrease was noted which was 1.5-fold higher in group II than in I. The increased formation period resulted from a prolonged inactive period in group II. These results point out a stimulatory effect of fluoride on the birth rate of osteoblasts. However, fluoride prolonged the lifespan of osteoblasts that had reduced activity.

Biochemical findings in blood of aged patients with femoral neck fractures: a contribution to the detection of occult osteomalacia.

A study was made in Geneva of 44 patients with femoral neck fractures and no risk factor of osteomalacia to determine concentrations of 25OHD3, calcium, phosphorus, alkaline phosphatase, albumin, and globulins in blood. The results were compared with those obtained for control groups of adult and aged subjects, as well as of 21 subjects operated on for hip osteoarthritis. For the detection of occult osteomalacia, femoral head bone tissue from 14 patients with fractures was examined by histomorphometric methods. In more than a third of the cases, 25OHD3 and serum albumin values were lower than those found for adult and even aged control subjects. Some slight histological signs of osteomalacia were observed in 1 patient with a femoral neck fracture, but there was no correlation between the histologic and the blood data. In practice, when dealing with aged people with no obvious risk of osteomalacia, it is important that a systematic antiosteomalacia treatment consisting of large supplements of vitamin D be avoided and that the conditions of diet and living be carefully controlled.