1,25-Dihydroxyvitamin D3 and the immune system

There is substantial evidence that lymphocytes and monocytes are targets for the actions of the hormonal form of vitamin D, 1,25-(OH)2D3 and that 1,25-(OH)2D3 acts to modulate the proliferation, differentiation, and immune functions of these cells. The effects of the hormone on lymphocytes are mediated directly as well as indirectly via the accessory monocytes. Depending upon the presence or absence of monocytes and the mode of lymphocyte activation, 1,25-(OH)2D3 can either stimulate or suppress lymphocytes. This evidence as well as clinical information and in vivo studies support a role of 1,25-(OH)2D3 in immunobiology. The physiologic, pathophysiologic, and pharmacologic implications of the immunomodulating properties of 1,25-(OH)2D3 however have not been well established.     

1,25-Dihydroxyvitamin D3 inhibits the clonogenic growth of transformed cells via its receptor

Anchorage-independent growth in soft agar is a unique property of transformed cells which is known to be correlated with tumorigenicity. We report here that 1,25-dihydroxyvitamin D3 suppresses colony formation by a number of cultured cancer cell lines in soft agar in a dose dependent manner with an ID50 of 5-7 X 10(-10) M. This effect is also achieved with analogues of 1,25-dihydroxyvitamin D3 in accordance with their binding affinity for the hormone's receptor. Only cells with 1,25-dihydroxyvitamin D3 receptor protein are inhibited in their colony formation by vitamin D analogs indicating that the hormone receptor complex may be integrally involved in the in vitro suppression of the anchorage-independent phenotype.     

DNA damage response pathway in radioadaptive response

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to the repair of DSBs and implicated to the current controversial observations on the expression of adaptive response.     

1,25-Dihydroxyvitamin D3 receptor replenishment in the chicken intestine

1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors.     

1,25-Dihydroxyvitamin D3 and the regulation of human cancer cell replication

Several human and animal cancer cell lines have been shown to possess specific high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The replication of several of these cell types has also been shown to be regulated by this hormone, both in vitro and in vivo. To further understand the mechanisms of these actions, we have examined cancer cells in vitro and in vivo. The in vitro studies extend our previous reports on the treatment of human breast cancer cells (T 47D) with 10(-9) to 10(-6) M 1,25-(OH)2D3, which resulted in a dose- and time-dependent decrease in cell numbers over 6 days. Treatment with 10(-8) M 1,25-(OH)2D3, which reduced cell numbers to approximately one half of those found in control cultures at 6 days, was associated with a doubling of the proportion of cells in the G2 + M phase of the cell cycle and was accompanied by a significant decline in the proportion of G0/G1 cells. At higher concentrations there was a significant decline in S phase cells with accumulation of cells in both G0/G1 and G2 + M phases. The antiestrogen, tamoxifen, at a concentration which caused similar effects on cell number, resulted in proportional decreases in both S and G2 + M phase cells and accumulation of G0/G1 cells. The effects of 1,25-(OH)2D3 on T 47D cell proliferation were associated with time- and concentration-dependent reductions in epidermal growth factor receptor levels to a minimum level of about half that seen in control cultures. The in vivo experiments extend our previous studies, which demonstrated marked inhibition of the growth of human cancer xenografts in immunosuppressed mice by 1,25-(OH)2D3. Xenograft growth was inhibited with 1,25-(OH)2D3 (0.1 microgram ip three times per week) but growth was rapidly restored when the 1,25-(OH)2D3 was withdrawn. Thus, there are clear-cut time- and dose-dependent, yet reversible, effects of 1,25-(OH)2D3 on the replication of human cancer cells in vitro and in vivo, which are possibly mediated through changes in growth factor receptor levels. Further study of these effects may advance understanding of the hormonal control of cellular replication in human cancers.     

Variation in DNA damage response pathway activity

Comment on: Kabacik S, et al. Cell Cycle 2011; 10:1152-61.     

mTORC1 pathway in DNA damage response

Living organisms have evolved various mechanisms to control their metabolism and response to various stresses, allowing them to survive and grow in different environments. In eukaryotes, the highly conserved mechanistic target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cellular metabolism, proliferation and survival. A growing body of evidence indicates that mTOR signaling is closely related to another cellular protection mechanism, the DNA damage response (DDR). Many factors important for the DDR are also involved in the mTOR pathway. In this review, we discuss how these two pathways communicate to ensure an efficient protection of the cell against metabolic and genotoxic stresses. We also describe how anticancer therapies benefit from simultaneous targeting of the DDR and mTOR pathways.     

Factors Affecting the Distribution and Stability of Unoccupied 1,25-Dihydroxyvitamin D3 Receptors

Abstract

Recently our laboratory reported that unoccupied 1,25(OH)₂D₃ receptors are found in the nuclei/chromatin fraction under low salt conditions in vitro (J. Biol. Chem. 255, 6799–6805, 1980). Additionally, various conditions exert differential effects on receptor solubilization and stability in vitro, potentially leading to confusion when these effects are not fully appreciated. In order to facilitate future studies, we have compiled a review of these factors and their effects. The conditions discussed include dilution, ionic strength (KCl and molybdate), protease inhibitors (Trasylol, PMSF and TPCK), temperature, ligand, glycerol, dithiothreitol, and EDTA.     

Monoubiquitylation in the Fanconi anemia DNA damage response pathway

The hereditary genetic disorder Fanconi anemia (FA) belongs to the heterogeneous group of diseases associated with defective DNA damage repair. Recently, several reviews have discussed the FA pathway and its molecular players in the context of genome maintenance and tumor suppression mechanisms [H. Joenje, K.J. Patel, The emerging genetic and molecular basis of Fanconi anaemia, Nat. Rev. Genet. 2 (2001) 446–457; W. Wang, Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins, Nat. Rev. Genet. 8 (2007) 735–748; L.J. Niedernhofer, A.S. Lalai, J.H. Hoeijmakers, Fanconi anemia (cross)linked to DNA repair, Cell 123 (2005) 1191–1198; K.J. Patel, Fanconi anemia and breast cancer susceptibility, Nat. Genet. 39 (2007) 142–143]. This review assesses the influence of post-translational modification by ubiquitin. We review and extract the key features of the enzymatic cascade required for the monoubiquitylation of the FANCD2/FANCI complex and attempt to include recent findings into a coherent mechanism. As this part of the FA pathway is still far from fully understood, we raise several points that must be addressed in future studies.     

25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 Promote the Differentiation of Human Subcutaneous Preadipocytes

1,25(OH)₂D₃ inhibits adipogenesis in mouse 3T3-L1 adipocytes, but little is known about its effects or local metabolism in human adipose tissue. We showed that vitamin D receptor (VDR) and 1α-hydroxylase (CYP27B1), the enzyme that activates 25(OH)D₃ to 1,25(OH)₂D₃, were expressed in human adipose tissues, primary preadipocytes and newly-differentiated adipocytes. Preadipocytes and newly-differentiated adipocytes were responsive to 1,25(OH)₂D₃, as indicated by a markedly increased expression of CYP24A1, a primary VDR target. 1,25(OH)₂D₃ enhanced adipogenesis as determined by increased expression of adipogenic markers and triglyceride accumulation (50% to 150%). The magnitude of the effect was greater in the absence of thiazolidinediones. 1,25(OH)₂D₃ was equally effective when added after the removal of differentiation cocktail on day 3, but it had no effect when added only during the induction period (day 0–3), suggesting that 1,25(OH)₂D₃ promoted maturation. 25(OH)D₃ also stimulated CYP24A1 expression and adipogenesis, most likely through its conversion to 1,25(OH)₂D₃. Consistent with this possibility, incubation of preadipocytes with 25(OH)D₃ led to 1,25(OH)₂D₃ accumulation in the media. 1,25(OH)₂D₃ also enhanced adipogenesis in primary mouse preadipocytes. We conclude that vitamin D status may regulate human adipose tissue growth and remodeling.     

1,25-Dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein

1,25-dihydroxyvitamin D₃ increases serum levels of bone Gla protein (BGP). The maximal increase occurs 12 h after injection and is given by 350 ng 1,25(OH)₂D₃ per 180 g body weight. In both 2 and 11 month-old male rats, the maximal increase is about 3 times the normal level, while in 2 month old female rats, the maximal increase is 2 times the normal level. These effects of 1,25(OH)₂D₃ in rats parallel the previously described effects of the vitamin on BGP secretion by rat osteosarcoma cells in culture.BGP is the first bone-specific protein whose synthesis in animals is dramatically increased by 1,25(OH)₂D₃. The possible functions of BGP in the biological actions of 1,25(OH)₂D₃ on bone are discussed.     

1,25-Dihydroxyvitamin D3 induces in vivo the decidualization of rat endometrial cells

Intraluminal injection of female rats at Day 5 of pseudopregnancy with 10-500 ng 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) significantly increased the uterine weight and induced decidual reaction. This effect was observed as early as the 3rd day after 1,25-(OH)2D3 injection. It was detectable only in the injected left horn and not in the non-injected right horn. A 500 ng dose of 25-(OH)D3 had no such effect. The present in-vivo results suggest that 1,25-(OH)2D3 may play a physiological role in endometrial cell differentiation into decidual cells, a crucial step in the process of blastocyst implantation.     

Open, repair and close again: chromatin dynamics and the response to UV-induced DNA damage

Due to its link with human pathologies, including cancer, the mechanism of Nucleotide Excision Repair (NER) has been extensively studied. Most of the pathway and players have been defined using in vitro reconstitution experiments. However, in vivo, the NER machinery must deal with the presence of organized chromatin, which in some regions, such as heterochromatin, is highly condensed but still susceptible to DNA damage. A series of events involving different chromatin-remodeling factors and histone-modifying enzymes target chromatin regions that contain DNA lesions. CPDs change the structure of the nucleosome, allowing access to factors that can recognize the lesion. Next, DDB1-DDB2 protein complexes, which mono-ubiquitinate histones H2A, H3, and H4, recognize nucleosomes containing DNA lesions. The ubiquitinated nucleosome facilitates the recruitment of ATP-dependent chromatin-remodeling factors and the XPC-HR23B-Centrin 2 complex to the target region. Different ATP-dependent chromatin-remodeling factors, such as SWI/SNF and INO80, have been identified as having roles in the UV irradiation response prior to the action of the NER machinery. Subsequently, remodeling of the nucleosome allows enzymatic reactions by histone-modifying factors that may acetylate, methylate or demethylate specific histone residues. Intriguingly, some of these histone modifications are dependent on p53. These histone modifications and the remodeling of the nucleosome allow the entrance of TFIIH, XPC and other NER factors that remove the damaged strand; then, gap-filling DNA synthesis and ligation reactions are carried out after excision of the oligonucleotide with the lesion. Finally, after DNA repair, the initial chromatin structure has to be reestablished. Therefore, factors that modulate chromatin dynamics contribute to the NER mechanism, and they are significant in the future design of treatments for human pathologies related to genome instability and the appearance of drug-resistant tumors.