Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.     

Evaluation of spermatogenic response of mice to the induction of mutations by combined treatment with X rays and antineoplastic drugs

Combined treatment with low doses of X-rays plus cyclophosphamide (0.25 Gy+25 mg/kg body weight) or X-rays plus mitomycin C (0.25 Gy+1.75 mg/kg body weight) did not induce significant dominant lethal effects in any stage of spermatogenesis when a parameter representing pre- and postimplantation loss, such as the decrease of live implants per female, was applied. After combined exposure to high doses of X-rays plus cyclophosphamide (1.00 Gy+100 mg/kg body weight) an increase of dominant lethal mutations (DLMs) was observed in differentiating spermatogonia, spermatids, and spermatozoa with the same parameter. Combined treatment with high doses of X-rays plus mitomycin C (1.00 Gy+5.25 mg/kg body weight) produced DLMs in differentiating spermatogonia and late spermatocytes. A calculation of enhanced risk was applied to the data of DLMs from the combined treatment regimen and was based on the proportion of dead implants (postimplantation loss only). Enhanced risk could be shown not only after high but also after low combined exposure to X-rays plus cyclophosphamide and X-rays plus mitomycin C. With low doses this enhanced risk was observed in spermatids for X-rays plus cyclophosphamide and in differentiating spermatogonia to early spermatocytes for X-rays plus mitomycin C.     

Effects of a combined treatment with X-rays and phenols on preimplantation mouse embryos in vitro

Summary Phenols are found everywhere in the environment. Therefore, the investigation of possible interactions between phenols and radiation is of some interest.The effects of a combination of X-rays and phenols (phenol itself and p-nitrophenol) were measured by the preimplantation mouse embryo-system in vitro. The microscopic visible development up to 144 h post conceptionem (h.p.c.), the number of cell nuclei, the DNA-content of each nucleus, the mitotic index, the labelling index, and the number of micronuclei were determined.There was not any indication that the effect of the irradiation was enhanced in a synergistic manner by the presence of phenols. All parameters measured lead to the conclusion that the effects of phenols plus X-rays are, at most, additive.     

The cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

In the CBA mouse testis about 10% of the stem cell population is highly resistant to neutron irradiation (D0, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea it appeared that in this cycle the S-phase is less radiosensitive (D0, 0.43 Gy) than the other phases of the cell cycle (D0, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most.     

Interaction between radiation and cadmium or mercury in mouse embryos during organogenesis

The response of mouse embryos to different concentrations of cadmium or mercury with or without low LET radiation was measured in terms of gross morphological anomalies and cellular changes. Single doses of the heavy metals (2 mg/kg body weight) were injected i.p. on day 8 of gestation 30 min before whole-body irradiation. Combined exposures to CdCl2 and X-rays led to a significant reduction in the rate of exencephaly compared with the high frequency after cadmium alone. The hypothesis that metallothionein, a sulphur-rich and metal-binding protein, may be responsible for the antagonism observed could not be confirmed. Mercuric chloride alone induced a low rate of exencephaly and the data on combined treatment suggest additivity with 0.5 and 1.0 Gy X-rays. Regarding cellular criteria, cell death in the eye anlage on day 9 of gestation was significantly suppressed after 0.5 Gy and especially after CdCl2 plus 0.5 Gy compared to cadmium alone. It is assumed that the reduction of cell lethality is correlated with the low occurrence of exencephaly. Concerning the proliferation of neuroblasts, cadmium stimulated the mitotic activity whereas X-rays depressed the proliferation capacity. After the combined treatment a distinct antimitotic effect was established.     

Combined action of X-rays and nonylphenol on mouse sperm

The aim of this study was to assess the effects of 2-weeks’ X-ray and/or nonylphenol (NP) exposure on male mice’s sperm count and quality. Pzh:SFIS mice were exposed to X-rays (0.05 Gy, 0.10 Gy, 0.20 Gy) or to nonylphenol (25 mg/kg bw, 50 mg/kg bw, 100 mg/kg bw) or to both agents (0.05 Gy + 25 mg/kg bw NP, 0.10 Gy + 50 mg/kg bw NP). At 24 h and 5 weeks after the end of exposure the sperm count, morphology and frequency of DNA damage in the male germ cells were estimated. Each agent alone diminished sperm count and morphology. The dose of 0.05 Gy of X-rays decreased the frequency of DNA damage. Combined exposure to lower doses of both agents significantly improved sperm morphology and decreased the level of DNA damage compared to one agent alone. Combined exposure to higher doses reduced the frequency of DNA damage compared to the effect of the appropriate dose of NP. Results of combined exposure to low doses of both agents suggest that 0.05 Gy of X-rays stimulate the DNA damagecontrol system and in consequence repair of DNA caused by X-rays and NP. It may be correlated with increased antioxidant capacity.     

RPA foci are associated with cell death after irradiation

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672-679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4-8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2-4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.     

Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6. II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and alpha-polymerase

The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase alpha, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 greater than xrs 5 greater than xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microcinematographic study of the lengthening of the cell cycle after x-ray and fast neutron irradiation

The median cycle duration of unirradiated EMT6 cells is equal to 9.68 +/- 0.20 h. It reaches 10.77 +/- 0.42 h, 12.95 +/- 0.62 h, 14.95 +/- 1.10 h, 16.15 +/- 1.87 h and 20.25 +/- 2.25 h after 250 kV X-rays doses of 1 Gy, 2 Gy, 4 Gy, 6 Gy and 8 Gy respectively and 11.23 +/- 1.03 h, 13.40 +/- 0.53 h and 15.13 +/- 1.15 h after p(65) + Be neutron doses of 0.75 Gy, 1.5 Gy and 2.5 Gy respectively. The corresponding mitotic delay (Tc irradiated - Tc controls) may be evaluated equal to congruent to 1.15 h per Gy for X-rays and to congruent to 2.30 h per Gy for neutrons.     

The individual and combined effects of X-irradiation and hyperthermia on early somite mouse embryos in culture.

The effects of 1) X-irradiation and 2) hyperthermia at a temperature of 43 degrees C individually and in combination have been investigated using cultured 8-day mouse embryos. B6C3F1 embryos were exposed to 0.3-2.0 Gy of X-rays, 5-20 min of heating, or 5 min of heating and irradiation at 0.3, 0.6, and 0.9 Gy. Irradiation alone at 0.3 Gy showed no apparent effect on embryonic development, but irradiation at 0.6-2.0 Gy caused a dose-dependent increase in malformed embryos. Heating alone for 5 min produced no malformed embryos, while heating for 10-20 min caused malformations as a function of heating time. Combined treatments produced higher frequencies (22.2-100%) of malformations than those of the sum of the separate treatments (0-41.7%). Malformations observed were primarily microphthalmia, microcephaly, and open neural tubes. The results indicate that in cultured mouse embryos irradiation combined with a "nonteratogenic dose" of hyperthermia directly exerts an additive effect on formation of the malformed embryos. In addition, a single occurrence of left-sided tail was produced by hyperthermia alone, while four occurrences were produced in combination with radiation.     

Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA. Transformation was observed only when exogenous DNA was still present during the heat shock. Shock fluid containing maltose-binding protein inhibited transformation (with 6 microgram of DNA per ml, half-maximal inhibition occurred at around 300 microgram of shock fluid per ml). DNA inhibited reconstitution (with 5 microgram of shock fluid per ml, half-maximal inhibition occurred at around 3 mg of DNA per ml).     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.     

Exposure to power frequency magnetic fields suppresses X-ray-induced apoptosis transiently in Ku80-deficient xrs5 cells

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser(15)), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G(1) cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression.     

In vitro study of the influence of donor age, nature of mitogen and exposure to X-rays on the proliferation rate of human lymphocytes

Proliferation of lymphocytes from donors belonging to 4 age groups (2, 25, 45 and 80 years) and exposed, in vitro, to 0 or 2 Gy of X-rays was studied in cultures stimulated by 3 different mitogens. Our results show that these 3 factors (age, irradiation and mitogen) play a role in the frequencies of cells observed in first, second, third or subsequent cell division in 48 or 72 h cultures.     

Effects of cisplatin and gamma-irradiation on cell survival, the induction of chromosomal aberrations and apoptosis in SW-1573 cells

PURPOSE: Cisplatin was found to radiosensitize SW-1573 cells by inhibition of PLDR. Therefore, it was investigated whether cisplatin combined with gamma-radiation leads to an increase in the number of chromosomal aberrations or apoptotic cells compared with radiation alone. METHODS: Confluent cultures of the human lung carcinoma cell line SW-1573 were treated with 1 microM cisplatin for 1 h, 4 Gy gamma-radiation, or a combination of both. Cell survival was studied by the clonogenic assay. Aberrations were analysed by FISH in prematurely condensed chromosomes (PCC) and the induction of apoptosis by counting fragmented nuclei. RESULTS: A radiosensitizing effect of cisplatin on cell survival was observed if time for PLDR was allowed. An increased number of chromosomal fragments were observed immediately after irradiation compared with 24 h after irradiation whereas color junctions are only formed 24 h after irradiation. No increase in chromosomal aberrations was found after combined treatment, but a significantly enhanced number of fragmented nuclei were observed when confluent cultures were replated after allowing PLDR. CONCLUSION: The inhibition of PLDR by cisplatin in delayed plated SW-1573 cells did not increase chromosomal aberrations, but increased the induction of apoptosis.     

Effect of magnesium on the growth and cell cycle of transformed and non-transformed epithelial rat liver cells in vitro

The effects of magnesium (Mg) restriction on cell growth and the cell cycle were determined in transformed (TRL-8) and non-transformed (TRL-12-15) epithelial-like rat liver cells. Cells were cultured in RPMI 1640 medium in which the Mg concentration was reduced to 0.5, 0.1, and 0 × the concentration in the regular RPMI 1640 media (100mg/l). Cell growth in the transformed cells was not influenced by the Mg restriction as greatly as in the non-transformed cell line. Transit through the cell cycle also exhibited an independence of the Mg in the medium in the transformed cells. When transformed cells were grown for two generations in Mg-limited medium, the growth rate slowed to a rate similar to that demonstrated by the non-transformed cells. Analysis by flow cytometry showed that transit through the cell cycle was minimally slowed in Mg deficient transformed cells; however, transit through the G₁ and S phases in the non-transformed cells was slowed. The TRL-8 cells in Mg-limited medium resulted in fewer nuclei in G₁ with subsequent increases in the percentages of S-phase nuclei. The TRL 12-15 cells reacted oppositely with the number of G₁ nuclei increased and the number of S-phase nuclei decreased. In respect to growth, these results show that epithelial cells respond in a similar manner to Mg-limitation as do fibroblast cells. The transformed cells exhibited a level of independence from Mg in respect to growth, reproduction, and cell-cycle kinetics.     

Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells

Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.     

Effects of X-irradiation on adventitious bud regeneration from in vitro leaf explants of Rosa hybrida

The effectiveness of X-radiation on regeneration of adventitious buds on in vitro leaf explants of three Rosa hybrida L. genotypes was studied. In vitro leaflet explants of roses produced adventitious buds when cultured in the dark for 1 week on Murashige and Skoog (MS) induction medium containing 6.8 μM thidiazuron (TDZ) + 0.49 μM indole-3-butyric acid (IBA) and subsequently transferred to MS regeneration medium containing 2.2 μM benzyladenine (BA) + 0.049 μM IBA in the presence of reduced light, at 15 μmol m-2 s-1 photosynthetically active radiation (PAR). Analysis of radiosensitivity by irradiating leaf explants with increasing doses of X-rays between 25 and 100 Gray (Gy) resulted in a decreasing rate of leaf explants regenerating buds from 47% to 0% respectively. The lethal dose for 50% of the regenerating explants (LD₅₀) in all the three genotypes was estimated to be 25 Gy at a dose rate 2 Gy/s. For the main experiment, doses of 5 and 15 Gy were selected and variations were observed between genotypes. Clone RUI 317 had the highest rate of adventitious bud regeneration, with 83.6% (2.5 buds/explant) at 5 Gy and 64% (1.8 buds/explant) at 15 Gy, compared to 89% (3.4 buds/explant) with the untreated control. Significant differences in the percentage of bud regeneration of the three genotypes were only observed at 15 Gy in comparison to the control and the number of buds formed per regenerating explant varied between 1 to 4. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression. However, the role of Tctex-1 in S-phase entry is dispensable in non-ciliated cells. Exogenously adding a phospho-mimic Tctex-1(T94E) mutant accelerates cilium disassembly and S-phase entry. These results support a model in which the cilia act as a brake to prevent cell cycle progression. Mechanistic studies show the involvement of actin dynamics in Tctex-1-regulated cilium resorption. Tctex-1 phosphorylated at Thr 94 is also selectively enriched at the ciliary transition zones of cortical neural progenitors, and has a key role in controlling G(1) length, cell cycle entry and fate determination of these cells during corticogenesis.     

Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp 70 Derived 14-Mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors on Peripheral Blood T Cells,B Cells and NKT Cells

Previous studies from Multhoff and colleagues reported that plasma membrane Hsp70 acts as a tumour-specific recognition structure for activated NK cells, and that the incubation of NK cells with Hsp70 and/or a 14-mer peptide derived from the N-terminal sequence of Hsp70 (TKDNNLLGRFELSG, TKD, aa 450–463) plus a low dose of IL-2 triggers NK cell proliferation and migration, and their capacity to kill cancer cells expressing membrane Hsp70. Herein, we have used flow cytometry to determine the influence of in vitro stimulation of peripheral blood mononuclear cells from healthy individuals with IL-2 or IL-15, either alone or in combination with TKD peptide on the cell surface expression of CD94, NK cell activatory receptors (CD16, NK2D, NKG2C, NKp30, NKp44, NKp46, NKp80, KIR2DL4, DNAM-1 and LAMP1) and NK cell inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2 and NKR-P1A) by CD3+CD56+ (NKT), CD3+CD4+, CD3+CD8+ and CD19+ populations. NKG2D, DNAM-1, LAMP1 and NKR-P1A expression was upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD in NKT, CD8+ T cells and B cells. CD94 was upregulated in NKT and CD8+ T cells. Concurrently, an increase in a number of CD8+ T cells expressing LIR1/ILT-2 and CD4+ T cells positive for NKR-P1A was observed. The proportion of CD8+ T cells that expressed NKG2D was higher after IL-2/TKD treatment, when compared with IL-2 treatment alone. In comparison with IL-15 alone, IL-15/TKD treatment increased the proportion of NKT cells that were positive for CD94, LAMP1 and NKRP-1A. The more potent effect of IL-15/TKD on cell surface expression of NKG2D, LIR1/ILT-2 and NKRP-1A was observed in B cells compared with IL-15 alone. However, this increase was not of statistical significance. IL-2/TKD induced significant upregulation of LAMP1 in CD8+ T cells compared with IL-2 alone. Besides NK cells, other immunocompetent cells present within the fraction of peripheral blood mononuclear cells were influenced by the treatment with low-dose interleukins themselves or in combination with hsp70 derived (TKD) peptide.