Characterization of cells expressing lipocalin-2 (LCN2) as a reporter.

Lipocalin-2 (LCN2), an effector molecule of the innate immune system that is small enough to be tagged as a reporter molecule, can be coupled with the ferric ion through a siderophore such as enterobactin (Ent). Mintbody (modification-specific intracellular antibody) can track a posttranslational protein modification in epigenetics. We constructed plasmids expressing the LCN2 hybrid of mintbody to examine the potential of LCN2 as a novel reporter for magnetic resonance imaging (MRI). Cells expressing the LCN2 hybrid of mintbody showed proper expression and localization of the hybrid and responded reasonably to Ent, suggesting their potential for in vivo study by MRI.

Life of the B10 mouse: a view from the hair follicles and tissue stem cells.

In our series of studies, the changes in the skin characteristics of mice caused by aging were investigated in correlation with the stem cells for keratinocytes and melanocytes in the natural hair cycle until middle age. The aim of the present review was to investigate these characteristics of hair follicles (HFs) at older age and complete the analysis of these changes as a study throughout the mouse lifetime. In addition, stem cells for keratinocytes and melanocytes were evaluated for changes in skin characteristics caused by aging. Postnatal day 200 (P200) appears to be the age of complete maturation of skin and the onset of aging with regard to HFs. Keratin 15-positive keratinocyte stem cells complete their localization as a quantitatively sufficient amount of progenitor in the hair bulge region and orchestrate the regeneration of hairs in every anagen phase thereafter. Although their frequency is low, an unusual structure of HFs, curved HFs, appear for the first time at P200. Thereafter, abnormal hair curvature continues to increase throughout life. In contrast, HF characteristics derived from melanocytes begin to show a high frequency of hypopigmented hair bulbs at P200 and appear to lead to a significant increase in the number of white hairs. Curved HFs and white hairs were considered biomarkers of aging in mice. The number of tyrosinase-related protein 2-positive melanocyte stem cells in the hair bulge is extremely low and may be one cause underlying not only the induction of melanocyte-derived characteristics by aging but possibly also that of keratinocyte-derived characteristics. These results provide insight into the mechanisms of the actions of stem cells on hair regeneration through the aging process.

Characterization of cells expressing MRI reporters for the analysis of epigenetics.

Epigenetics is thought to be involved in highly advanced life phenomena, and its regulation has created new opportunities in regenerative medicine. Mintbody (modification-specific intracellular antibody) can track a posttranslational protein modification in epigenetics using a genetic system encoded within a single chain of variable fragments tagged with a fluorescent protein. Magnetic resonance imaging (MRI) is a technique that allows observation of specific molecules in living organisms. The ferritin heavy chain (FTH1) is one of the MRI reporters used in mammals. The combination of FTH1 with mintbody may show remarkable ability as a reporter for MRI to investigate epigenetics in the deep part of a living organism. This article discusses the suitability and safety of FTH1 for use in the analysis of epigenetics by MRI. Cells expressing the FTH1 hybrid of mintbody showed insufficiently increased sensitivity by MRI even in the presence of excess iron. After incubation with ferric ammonium citrate, DNA damage was found in cells expressing the FTH1 hybrid of mintbody. The use of FTH1 as a genetically encoded reporter for MRI appears to be limited by the requirement of metal and its relatively low sensitivity. These results suggest future directionality and the possibility of studying epigenetics in vivo.

Changes in characteristics of murine hair follicles and tissue stem cells by aging.

The aging process is closely related to the organization of stem cells, and skin is thought to be one of the suitable models for its investigation. We have focused on the murine hair follicle to verify this idea because it shows typical aging phenotypes and is a self-renewing structure reconstituted by its own stem cells. However, how changes in the characteristics of the hair follicle and in the behavior of tissue stem cells in the natural hair cycle occur are not fully understood. We investigated the number, morphology and pigmentation of hair follicles in anagen phases during the aging process. In addition, stem cells for keratinocytes and melanocytes were examined to evaluate the correlation between changes in skin characteristics and the stem cells. The remarkable changes caused by aging appeared to be the significant increase in qualitative phenotypes such as curved hair follicles and white hair. A significant difference between the number of keratinocyte and melanocyte stem cells in the hair bulge region is likely to be involved in these changes. Our findings may be important for understanding the mechanisms of the actions of stem cells on hair regeneration and for clarifying the mechanisms of age-related phenotypes.

Menadione/Ascorbate Induces Overproduction of Mitochondrial Superoxide and Impairs Mitochondrial Function in Cancer: Comparative Study on Cancer and Normal Cells of the Same Origin.

BACKGROUND/AIM: The menadione/ascorbate (M/A) combination has attracted attention due to the unusual ability of pro-vitamin/vitamin combination to kill cancer cells without affecting the viability of normal cells. The aim of this study was to elucidate the role of M/A in targeting cancerous mitochondria. MATERIALS AND METHODS: Several cancer and normal cell lines of the same origin were used. Cells were treated with different concentrations of M/A for 24 h. The cell viability, mitochondrial superoxide, mitochondrial membrane potential, and succinate were analyzed using conventional analytical tests. RESULTS: M/A exhibited a highly specific suppression on cancer cell growth and viability, without adversely affecting the viability of normal cells at concentrations attainable by oral or parenteral administration in vivo. This effect was accompanied by: (i) an extremely high production of mitochondrial superoxide in cancer cells, but not in normal cells; (ii) a significant dose-dependent depolarization of mitochondrial membrane and depletion of oncometabolite succinate in cancer cells. CONCLUSION: The anticancer effect of M/A is related to the induction of severe mitochondrial oxidative stress in cancer cells only. Thus, M/A has a potential to increase the sensitivity and vulnerability of cancer cells to conventional anticancer therapy and immune system.

Chromosome instability caused by mutations in the genes involved in transcription and splicing.

Mutations in molecules involved in transcription and splicing can cause chromosome instability such as sister chromatid exchanges. We isolated and characterized responsible genes from mammalian temperature-sensitive mutant cells showing chromosome instability. A mutation in the largest subunit of RNA polymerase II affected DNA synthesis in S phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges. The yeast mutant harboring a homologous mutation showed very similar phenotype to that of the mammalian mutant. A mutation in Smu1, which is involved in splicing, also affected DNA synthesis in S and G2 phase-arrested cells, resulting in abnormal induction of sister chromatid exchanges and chromosomal aberrations. These cells showed a connection between defects of RNA metabolism and induction of chromosome instability. Genome instability appeared to be caused by links between RNA metabolism and replication resulting in genomic recombination. RNA metabolism can be regarded as one possible driver of genome modification triggering genome evolution.

Let's think again about using mammalian temperature-sensitive mutants to investigate functional molecules-The perspectives from the studies on three mutants showing chromosome instability.

This review evaluates the use of temperature-sensitive (ts) mutants to investigate functional molecules in mammalian cells. A series of studies were performed in which mammalian cells expressing functional molecules were isolated from ts mutants using complementation by the introduction and expression of the responsible protein tagged with the green fluorescent protein. The results showed that chromosome instability and cell-cycle arrest were caused by ts defects in the following three molecules: the largest subunit of RNA polymerase II, a protein involved in splicing, and ubiquitin-activating enzyme. The cells expressing functional protein were then isolated by introducing the responsible gene tagged with the green fluorescent protein to complement the ts phenotype. These cells proved to be useful in analyzing the dynamics of RNA polymerase II in living cells. Analyses of the functional interaction between proteins involved in splicing were also useful in the investigation of ts mutants and their derivatives. In addition, these cells demonstrated the functional localization of ubiquitin-activating enzyme in the nucleus. Mammalian ts mutants continue to show great potential to aid in understanding the functions of the essential molecules in cells. Therefore, it is highly important that studies on the identification and characterization of the genes responsible for the phenotype of a mutant are carried out.

Effects of gamma rays on the regeneration of murine hair follicles in the natural hair cycle.

PURPOSE: This review evaluates the effects of γ-rays on the regeneration of murine hair follicles in the natural hair cycle. A series of studies were performed to investigate this issue, in which the whole bodies of C57BL/10JHir mice in the 1st telogen phase of the hair cycle were irradiated with γ-rays. RESULTS: The dermis of the irradiated skin showed a decrease in hair follicle density and induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs in the 2nd and 3rd anagen phases. An increased frequency of hypopigmented hair bulbs was still observed in the later hair cycle at postnatal day 200. There was no significant difference in the number of stem cells in the hair bulge region between control and irradiated skin. CONCLUSIONS: These results show that the effects of γ-rays on the pigmentation of murine hair follicles are persistently carried over to later hair cycles, although those on the number and structure of hair follicles appear to be hidden by the effects of aging. Our findings may be important for understanding the mechanisms of the actions of stem cells on hair regeneration in connection with age-related phenotypes.

Regeneration of Murine Hair Follicles is Inhibited by Low-Dose-Rate Gamma Irradiation.

To determine whether the effects of low-dose-rate gamma (γ) irradiation are identifiable in the regeneration of murine hair follicles, we irradiated whole bodies of C57BL/10JHir mice in the first telogen phase of the hair cycle with 137Cs γ-rays. The mice were examined for effects on hair follicles, including number, morphology, and pigmentation in the second anagen phase. Effects of γ-radiation on melanocyte stem cells were also investigated by the indirect immunolabeling of tyrosinase-related protein 2 (TRP2). Irradiated skin showed a decrease in hair follicle density and the induction of curved hair follicles along with the presence of white hairs and hypopigmented hair bulbs. There was a small, but not significant, change in the number of TRP2-positive melanocyte stem cells in the hair bulge region of the irradiated skin. These results suggest that low-dose rate γ-irradiation does not deplete melanocyte stem cells, but can damage stem cells and progenitors for both keratinocytes and melanocytes, thereby affecting the structure and pigmentation of regenerated hair follicles in the 2nd anagen phase.

The effects of gamma rays on the regeneration of hair follicles are carried over to later hair cycles.

PURPOSE: The purpose of this study was to determine whether the effects of gamma rays on the regeneration of hair follicles are carried over to later hair cycles. MATERIALS AND METHODS: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with (60)Co γ-rays. Mice were examined for the effects on hair follicles, including their number, morphology and pigmentation in the 3rd anagen phase. Effects of γ-rays on hair follicle stem cells were investigated by the indirect immunolabeling of keratin 15 (K15). RESULTS: Decreased hair follicle density and induction of curved hair follicles were observed in the dermis of irradiated skin. In addition, white hair and hypopigmented hair bulbs were found. The number of K15-positive hair follicle stem cells in the hair bulge region of irradiated skin appeared to decrease slightly but not significantly. CONCLUSIONS: These results suggest that the effects of γ-rays are carried over to a later hair cycle to affect the number, structure and pigmentation of hair follicles in the 3rd anagen phase when stem cells and committed progenitors for keratinocytes and melanocytes are irradiated in the 1st telogen phase.

Nuclear localization of ubiquitin-activating enzyme Uba1 is characterized in its mammalian temperature-sensitive mutant.

In our previous study, a point mutation in Uba1, the gene encoding ubiquitin-activating enzyme, was identified in temperature-sensitive (ts) CHO-K1 mutant tsTM3 cells, which led to a Met-to-Ile substitution at amino acid 256 in Uba1 protein. Characterization of this mutant revealed a deficiency of nuclear Uba1 and impaired ubiquitination in the nucleus. The ts defects in tsTM3 were complemented by the expression of the wild-type Uba1 tagged with green fluorescent protein (GFP). In this study, the expression and localization of Uba1 were investigated using the various forms of Uba1 tagged with GFP. Western blot analysis and confocal microscopy revealed that nuclear localization of Uba1, as well as even the modified and truncated forms of Uba1, appears to be essential to rescue tsTM3 cells. The localization of Uba1 in the nucleus, even if it was a small amount, was proportional to the efficiency of complementation of tsTM3 cells. Uba1 plays an important role in the nucleus, and a ts mutation found in tsTM3 cells appears to result in the loss of localization of Uba1 in the nucleus.

Analysis of a temperature-sensitive mutation in Uba1: Effects of the click reaction on subsequent immunolabeling of proteins involved in DNA replication.

In our previous study, a Met-to-Ile substitution at amino acid 256 in the catalytic domain of Uba1 was determined in temperature-sensitive CHO-K1 mutant tsTM3 cells, which exhibited chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 °C. Mutant cells were also characterized by a significant decrease of Uba1 in the nucleus with decreased ubiquitination activity at 39 °C. Defects of ubiquitination activity in the nucleus resulted in an inappropriate balance between Cdt1 and geminin, a licensing factor of DNA replication and its inhibitor. In the present study, we found that the Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction inhibits the subsequent indirect immunolabeling of Cdt1 but allows for the detection of PCNA with nascent DNA. Using a procedure without the click reaction, we also demonstrated that Cdt1 remained close to active replication sites in tsTM3 cells at the nonpermissive temperature. Analysis of genome replication by DNA fiber spreading revealed that DNA synthesis continues for at least 10 h after incubation at 39 °C, suggesting that impaired ubiquitination in the nucleus, caused by the defect of Uba1, affected DNA replication only after a long delay.

Characterization of ubiquitin-activating enzyme Uba1 in the nucleus by its mammalian temperature-sensitive mutant.

Temperature-sensitive (ts) CHO-K1 mutant tsTM3 exhibits chromosomal instability and cell-cycle arrest in the S to G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. Previously, complementation tests with other mutants showed that tsTM3 harbors a genetic defect in the ubiquitin-activating enzyme Uba1. Sequence comparison of the Uba1 gene between wild-type and mutant cells in this study revealed that the mutant phenotype is caused by a G-to-A transition that yields a Met-to-Ile substitution at position 256 in hamster Uba1. The ts defects in tsTM3 were complemented by expression of the wild-type Uba1 tagged with green fluorescent protein. Expression of the Uba1 primarily in the nucleus appeared to rescue tsTM3 cells. Incubation at 39°C resulted in a decrease of nuclear Uba1 in tsTM3 cells, suggesting that loss of Uba1 in the nucleus may lead to the ts defects. Analyses with the fluorescent ubiquitination-based cell cycle indicator revealed that loss of function of Uba1 leads to failure of the ubiquitin system in the nucleus. Incubation at 39°C caused an increase in endogenous geminin in tsTM3 cells. A ts mutation of Uba1 found in tsTM3 cells appears to be a novel mutation reflecting the important roles of Uba1 in nucleus.

Exposure to gamma-rays at the telogen phase of the hair cycle inhibits hair follicle regeneration at the anagen phase in mice.

PURPOSE: The effects of ionizing radiations on somatic stem cells largely remain to be studied. Hair follicles are self-renewing structures that reconstitute themselves throughout the hair cycle, which is comprised of the following phases: Anagen (growth), catagen (regression) and telogen (resting), suggesting the presence of their own stem cells. MATERIALS AND METHODS: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with γ-rays. Mice were examined for effects on hair follicles, including their number, morphology and pigmentation in the 2nd anagen phase. RESULTS: Decreased hair follicle density and induction of curved hair follicles were observed in the dermal skin of irradiated mice. In addition to these keratinocyte-derived anomalies, melanocyte-derived anomalies including white hair and hypopigmented hair bulbs were found. The decrease in hair follicle density and the increase in the frequency of hypopigmented hair bulbs were dependent on the dose of γ-rays. CONCLUSIONS: These results suggest that γ-rays damage stem cells and progenitors for keratinocytes and melanocytes, thereby affecting the structure and character of regenerated hair follicles. The density of hair follicles and pigment production in hair bulbs are established as criteria for the effects of γ-rays on the hair cycle.

Effects of low-dose heavy ions on embryonic development in mice and on melanocyte differentiation in the epidermis and hair bulb.

The effects of prenatal low-dose irradiation with heavy ions on embryonic development in mice and on melanocyte differentiation are not well understood. We performed whole-body irradiation of pregnant C57BL/10J mice at embryonic Day 9 (E9) with a single dose of γ-rays, silicon, argon or iron ions. The number of living embryos and embryonic body weight at E18 decreased after exposure to heavy ions at high doses. Malformations such as small eyes and limb anomalies were observed in heavy-ion-treated embryos, but not in γ-ray-treated embryos. The frequency of abnormally curved tails was increased by exposure to γ-rays and argon and iron ions even at a dose of 0.1 Gy (P < 0.05). In contrast, a dose-dependent decrease in the number of epidermal melanoblasts/melanocytes and hair bulb melanocytes was observed after 0.1 Gy irradiation with γ-rays or heavy ions (P < 0.01). The decrease in the number of dorsal hair bulb melanocytes, dorsal and ventral epidermal melanoblasts/melanocytes and ventral hair bulb melanocytes was not necessarily correlated with the linear energy transfer of the radiation tested. Moreover, the effects of heavy ions were larger on the ventral skin than on the dorsal skin, indicating that the sensitivity of melanocytes to heavy ions differs between the dorsal and ventral skin. Taken together, these results suggest that the effects of the low-dose heavy ions differ between cell types and tissues, and the effects on the prenatal development of mice and melanocyte development are not necessarily greater than those of γ-rays.

Effects of low-dose γ-rays on the embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs.

The effects of low-dose γ-rays on the embryonic development of animal cells are not well studied. The mouse melanocyte is a good model to study the effects of low-dose γ-rays on the development of animal cells, as it possesses visible pigment (melanin) as a differentiation marker. The aim of this study is to investigate in detail the effects of low-dose γ-rays on embryonic development of mouse melanoblasts and melanocytes in the epidermis and hair bulbs at cellular level. Pregnant females of C57BL/10J mice at nine days of gestation were whole-body irradiated with a single acute dose of γrays (0.1, 0.25, 0.5, and 0.75 Gy), and the effects of γ-rays were studied by scoring changes in the development of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes at 18 days in gestation. The number of epidermal melanoblasts and melanocytes, hair follicles, and hair bulb melanocytes in the dorsal and ventral skins was markedly decreased even at 0.1 Gy-treated embryos (P < 0.001), and gradually decreased as dose increased. The effects on the ventral skin were greater than those on the dorsal skin. The dramatic reduction in the number of melanocytes compared to melanoblasts was observed in the ventral skin, but not in the dorsal skin. These results suggest that low-dose γ-rays provoke the death of melanoblasts and melanocytes, or inhibit the proliferation and differentiation of melanoblasts and melanocytes, even at the low dose.

Enlargement of speckles of SF2/ASF due to loss of function of Smu1 is characterized in the mammalian temperature-sensitive mutant.

A temperature-sensitive (ts) CHO-K1 mutant cell line, tsTM18, exhibits chromosomal instability with decreased DNA synthesis at the nonpermissive temperature, 39 °C. An amino acid substitution in Smu1 underlying the ts phenotypes of tsTM18 cells was identified previously. We also found a ts defect in splicing of the unc52/perlecan gene. In the present study, we have generated cell lines expressing Smu1 tagged with green fluorescent protein (GFP) to study the dynamics of Smu1 in living cells. The hybrids complement deficiencies in tsTM18 cells and allow them to grow normally at 39 °C. GFP-tagged Smu1 is found in speckles in many discrete nucleoplasmic sites, and most of these also contained SF2/ASF. SF2/ASF is a member of the serine/arginine (SR)-rich splicing group of factors that are necessary for spliceosome assembly and can influence alternative splicing. SF2/ASF is also involved in the integrity of genome maintenance. In tsTM18 cells cultured at 39 °C, the Smu1 ts defect appears to alter SF2/ASF localization, suggesting a physiological association of Smu1 with SF2/ASF. The significant decrease of Smu1 may lead the enlargement of speckles of SF2/ASF. These data show the importance of Smu1 as a regulator of splicing and genome maintenance.

Effects of low-dose heavy ions on the postnatal development of mice and the yield of white spots in the mid-ventrum and tail-tips.

Effects of prenatal low-dose irradiations of heavy ions on the postnatal development of mice and of melanocytes have not been well studied. Pregnant females of C57BL/10J mice were irradiated whole-body at 9 days of gestation with a single acute dose of γ-rays, silicon (Si, 57 keV/µm), argon (Ar, 100 keV/µm) and iron (Fe, 220 keV/µm) ions. The effects were studied by scoring changes in the postnatal development of mice as well as in the pigmentation of cutaneous coats and tail-tips of their offspring 22 days after birth. The survival to day 22 decreased from the offspring exposed to 0.4 Gy of argon and iron ions and to 0.75 Gy of silicon ions. White spots were found in the mid-ventrum and tail-tips of irradiated offspring. The frequency and size of the white spots in the mid-ventrum in mice exposed to silicon, argon and iron ions were greater than those of γ-rays. Even in the low dose (0.1 Gy), γ-rays and heavy ions increased the frequency of the ventral spots. The RBE estimated by the frequency of the ventral spots was 2.3 (Si), 3.1 (Ar) and 4.5 (Fe). These results suggest that prenatal exposure to heavy ions possesses a greater effect on the postnatal development of mice as well as melanocyte development than does exposure to γ-rays.

Characterization of cells expressing RNA polymerase II tagged with green fluorescent protein: effect of ionizing irradiation on RNA synthesis.

PURPOSE: To isolate and to characterize cells expressing RNA polymerase II tagged with green fluorescent protein for analyses of the effects of ionizing radiation on transcription in living cells. MATERIALS AND METHODS: We introduced an alpha-amanitin-resistant mutation into a vector encoding the largest subunit of RNA polymerase II tagged with green fluorescent protein (GFP-pol). Cell lines stably expressing functional GFP-pol were isolated under selection with alpha-amanitin from a Chinese hamster cell line, CHO-K1, and a radiation-sensitive mutant CHO cell line, XR-1. RESULTS: We tested the functionality of the fusion protein in vivo by determining RNA synthesis activity by incorporation of nucleoside analogues. Both CHO-K1 and XR-1 cells expressing GFP-pol had properties similar to those of their respective parental cell lines, indicating that GFP-pol is functional. CONCLUSIONS: These stable lines might prove useful for analyses of the roles of transcription after ionizing radiation.

The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant.

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell-cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39 degrees C. We previously identified an amino acid substitution in Smu1 that underlies the temperature-sensitive phenotypes of tsTM18 cells. In the present study, we confirmed that Smu1 is associated with the temperature-sensitive defect of tsTM18 by RNA interference. We also found an early temperature effect in DNA synthesis. Because genetic studies of nematodes revealed that smu-1 is involved in splicing of the unc52/perlecan pre-mRNA, we analysed the perlecan transcript in tsTM18 cells by reverse transcription-polymerase chain reaction (RT-PCR). The perlecan PCR product amplified from RNA of tsTM18 cells cultured at 39 degrees C appeared to be a mixture of variants. Sequence analysis identified at least six variants that result from alternative splicing and intron retention. Comparison of the results of perlecan RT-PCR analysis with those of analysis of four other genes suggested that the splicing defect in the perlecan gene is unique and that it is conserved through evolution.