Gerald L. Chan

Ultraviolet light irradiation of defined-sequence DNA under conditions of chemical photosensitization

Abstract— The formation of cyclobutane pyrimidine dimers and UV light‐induced (6‐4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6‐4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.

Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light.

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhance of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells.

Progranulin Recruits HSP70 to β-Glucocerebrosidase and Is Therapeutic Against Gaucher Disease

Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of β-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

Association Between Progranulin and Gaucher Disease.

Background Gaucher disease (GD) is a genetic disease caused by mutations in the GBA1 gene which result in reduced enzymatic activity of β-glucocerebrosidase (GCase). This study identified the progranulin (PGRN) gene (GRN) as another gene associated with GD. Methods Serum levels of PGRN were measured from 115 GD patients and 99 healthy controls, whole GRN gene from 40 GD patients was sequenced, and the genotyping of 4 SNPs identified in GD patients was performed in 161 GD and 142 healthy control samples. Development of GD in PGRN-deficient mice was characterized, and the therapeutic effect of rPGRN on GD analyzed. Findings Serum PGRN levels were significantly lower in GD patients (96.65 ± 53.45 ng/ml) than those in healthy controls of the general population (164.99 ± 43.16 ng/ml, p < 0.0001) and of Ashkenazi Jews (150.64 ± 33.99 ng/ml, p < 0.0001). Four GRN gene SNPs, including rs4792937, rs78403836, rs850713, and rs5848, and three point mutations, were identified in a full-length GRN gene sequencing in 40 GD patients. Large scale SNP genotyping in 161 GD and 142 healthy controls was conducted and the four SNP sites have significantly higher frequency in GD patients. In addition, “aged” and challenged adult PGRN null mice develop GD-like phenotypes, including typical Gaucher-like cells in lung, spleen, and bone marrow. Moreover, lysosomes in PGRN KO mice exhibit a tubular-like appearance. PGRN is required for the lysosomal appearance of GCase and its deficiency leads to GCase accumulation in the cytoplasm. More importantly, recombinant PGRN is therapeutic in various animal models of GD and human fibroblasts from GD patients. Interpretation Our data demonstrates an unknown association between PGRN and GD and identifies PGRN as an essential factor for GCases lysosomal localization. These findings not only provide new insight into the pathogenesis of GD, but may also have implications for diagnosis and alternative targeted therapies for GD.

Study of Basal Cell Nevus Syndrome Fibroblasts after Treatment with DNA-Damaging Agents

Basal cell nevus syndrome (BCNS) is a rare autosomal dominant inherited disorder (1). About 20% of gene carriers develop brain tumors in early childhood. Basal cell nevus syndrome patients are abnormally susceptible to radiation-induced cancer; several patients treated with radio-therapeutic doses have developed large numbers of basal cell tumors in the irradiated field within 6 mo to 3 yr of ex posure (2-5). Featherstone et al. (6) reported that fibroblasts from BCNS patients showed no increased susceptibility to X-ray-induced cell killing; however, GO-irradiated lymphocytes from BCNS patients were found to have a significantly higher level of X-ray-induced chromosomal aberrations (CAs) compared with normal cells. On the other hand, Chan and Little (7) reported that fibroblasts from BCNS patients were slightly hypersensitive to X-ray-induced lethality.

Correlation of in vitro transformation with in vivo tumorigenicity in 10T1/2 mouse cells exposed to UV light.

ImagesFig.

Response of Plateau-phase Mouse Embryo Fibroblasts to Ultra-violet Light

Clonogenic survival was measured in plateau-phase cultures of the 10T1/2 mouse cell line exposed to 254 nm ultra-violet light. The survival curve was found to be biphasic, Do for the two components being 37 and 1191 erg/mm2 respectively. This extreme resistance at higher doses can only be partly accounted for by the increased cytoplasmic absorption of U.V.L. due to an increased thickness of plateau-phase cells. When the cultures were held for 24 hours in plateau phase in conditioned medium after irradiation, recovery yielding a 1.4-fold enhancement of survival was found at higher doses. This recovery process was inhibited by neither caffeine nor cycloheximide. When caffeine was given for 48 hours after sub-culture, the effect on survival was also negligible. We propose that this plateau-phase recovery process is associated with excision repair of DNA adducts induced by U.V.L. Delayed sub-culturing favours the excision mode of repair and renders the post-replication mode less necessary.

Cross-sensitivity of certain xeroderma pigmentosum and cockayne syndrome fibroblast strains to both ionizing radiation and ultraviolet light

It has been suggested that DNA adducts induced by a variety of agents may be classified as being either UV-like or x-ray-like, depending on the degree of similarity between the structural distortion introduced into the DNA helix by these adducts and that introduced by far ultraviolet light or x-rays (Regan and Setlow 1974; Cerutti 1975). While DNA adducts may fall into structurally related groups, it cannot be precluded that cellular repair pathways may be shared to deal with structurally diverse groups of adducts. This possibility has not been seriously entertained hitherto since the UV-sensitive xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells have not been shown to be cross-sensitive to x-rays (Arlett and Harcourt 1980; Wade and Chu 1979). Likewise, ataxia telangiectasia (AT) cells which are defective in x-ray repair have not been shown to be UVsensitive (Lehmann et al. 1977). We report here the identification of one strain each of XP and CS cells which are cross-sensitive to both UV and x-rays. The end point we have used is clonogenic survival of cultured diploid skin fibroblasts. The two XP strains, XP12BE belonging to complementation group A and XP8BE belonging to complementation group C were obtained from the American Type Culture Collection, Rockville, MD, USA. The two CS strains, GM1856 and GM1629, the AT strain GM2052, as well as the two control normal strains AG1518 and AG1522, were obtained from the Human Genetic Mutant Cell Repository, Camden, N J, USA. In these survival experiments, cells were plated in appropriate dilutions on to triplicate 10 cm diameter culture dishes, incubated overnight and exposed to 254 nm wavelength ultraviolet light or 220 kVp x-rays. After irradiation, cells were incubated for 2 3 weeks for the formation of macroscopic colonies. Details of our culture conditions, irradiation procedures and scoring of survivors have been described elsewhere (Chan and Little 1979; Weichselbaum et al. 1980). The results of our experiments are tabulated in Table 1. With the exception of XP8BE, the UV survival curves were biphasic. The two CS strains tested were sensitive to UV light to approximately the same extent as XP complementation group C strains, as judging by comparison of D O values (inverse of slope of exponential portion of survival curve) reported by us and others (Chan and Little 1979; Andrews et al. 1978). However, the GM1856 strain responded to x-ray killing as normal cells while the GM1629 strain responded as hypersensitive. Similarly,

On the Nature of Oncogenic Transformation of Cells

Publisher Summary This chapter presents a class of genes in the genome of higher cells, which is called “growth genes.” The existence of the growth genes in multiplicity in a genome is envisaged as necessitated by the participation of each growth gene in a distinct pathway or related pathways of differentiation. The primary function of growth genes is to elicit cell growth; they are also intricately involved in programs of gene expression, which operate during development of the organism. After transient expression during development, they become permanently repressed for the remainder of the organisms life cycle except in certain cases of tissue regeneration and cell renewal. Given the proper conditions, postdevelopmental expression of any growth gene in a cell can lead to oncogenic transformation. To consummate the oncogenic process, mutations alone do not appear to be sufficient. Additional steps involving genomic reprogramming are necessary.

Further Studies on the Survival of Non-proliferating Human Diploid Fibroblasts Irradiated with Ultraviolet Light

Labelling index data showed that in AG1518 cells, a diploid human fibroblast strain, there was a lag period of at least 14 hours between subculture from the density-inhibited plateau phase of growth and entry into DNA synthesis. Cells irradiated with 254 nm wavelength U.V. light 8 hours after subculture did not exhibit the same degree of resistance as cells irradiated in plateau phase and subcultured immediately. When cells were arrested from proliferation by maintenance in an arginine and glutamine deficient medium for 72 hours, they were nearly as resistant to U.V. light as plateau phase cells although maintenance in this medium for 24 hours after irradiation supported further recovery only after low U.V. doses. One strain of Cockayne syndrome fibroblasts was found to be resistant to U.V light in plateau phase while another strain was found to have the same survival response whether it was irradiated in the plateau or log phase of growth.