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.