CERKL is upregulated in cutaneous squamous cell carcinoma and maintains cellular sphingolipids and resistance to oxidative stress.

BACKGROUND: Ceramide kinase-like protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, quantitative polymerase chain reaction and immunohistochemistry. CERKL was knocked down in cSCC cells using small interfering RNA. Sphingolipid content was analysed by liquid chromatography-mass spectrometry. Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin V binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL is also expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

CERKL is Upregulated in Cutaneous Squamous Cell Carcinoma and Maintains Cellular Sphingolipids and Resistance to Oxidative Stress.

BACKGROUND: Ceramide Kinase-Like Protein (CERKL) was originally described in retinal tissue. CERKL has been shown to protect cells from oxidative stress, and mutations in CERKL underlie the inherited disease, retinitis pigmentosa. CERKL expression maintains cellular sphingolipids via an unknown mechanism. OBJECTIVES: To determine whether CERKL is expressed in epidermis and cutaneous squamous cell carcinoma (cSCC) and whether CERKL expression affects cSCC sphingolipid metabolism and susceptibility to oxidative stress. METHODS: CERKL expression was determined by RNA-Seq, qPCR and immunohistochemistry. CERKL was knocked down in cSCC cells using siRNA. Sphingolipid content was analyzed by liquid chromatography-mass spectrometry (LC-MS). Oxidative stress was induced by treatment with H2 O2 , and apoptosis was measured using flow cytometry to determine annexin v binding. RESULTS: CERKL mRNA and protein are highly expressed in actinic keratosis and cSCC in comparison with normal epidermis. CERKL also is expressed in metabolically active epithelial cells in normal hair bulbs and sebaceous glands. CERKL knockdown in cultured cSCC cells reduces cellular sphingolipid content and enhances susceptibility to oxidative stress. CONCLUSIONS: These findings suggest that CERKL may be important in cSCC progression and could lead to novel strategies for prevention and treatment of cSCC.

Inhibitors of poly(adenosine diphosphate-ribose) synthesis: effect on other metabolic processes.

3-Aminobenzamide and benzamide, purported to be specific inhibitors of the synthesis of poly(adenosine diphosphate-ribose), were used to elucidate possible functions of this biopolymer. These compounds, at frequently used experimental concentrations, not only inhibited the action of poly(adenosine diphosphate-ribose) synthetase but also affected cell viability, glucose metabolism, and DNA synthesis. Thus, the usefulness of 3-aminobenzamide and benzamide may be severely restricted by the difficulty of finding a dose small enough to inhibit the synthetase without producing additional metabolic effects.

Biological damage from intranuclear tritium: DNA strand breaks and their repair.

Isotopic decay in tritiated thymidine in the DNA of frozen (-196 degrees C) Chinese hamster cells causes breaks in DNA strands to accumulate at a rate of 2.1 breaks per decay. After DNA is thawed the tritium-induced breaks repair rapidly with a half-time of 15 minutes at 37 degrees C. In comparison to breakage by x-rays, the efficiency of DNA strand breakage by tritium is equivalent to 0.48 rad per decay. This dose per decay is close to that predicted by simple dosimetric considerations (0.38 rad per decay) for irradiation by the beta particles from tritium.

Distribution of chromatids at mitosis.

The distribution of labeled chromatids at the second mitosis after labeling with tritiated thymidine is random in both Vicia faba (the broad bean) and Potorous tridactylis (the rat kangaroo). This finding is contrary to that predicted by the hypothesis that chromatids containing "grandparent" polynucleotide templates segregate from those containing "parent" templates.

Pol eta is required for DNA replication during nucleotide deprivation by hydroxyurea.

Hydroxyurea reduces DNA replication by nucleotide deprivation, whereas UV damage generates DNA photoproducts that directly block replication fork progression. We show that the low fidelity class Y polymerase Pol eta is recruited to proliferating cell nuclear antigen at replication forks both by hydroxyurea and UV light. Under nucleotide deprivation, Pol eta allows cells to accumulate at the G1/S boundary by facilitating slow S-phase progression and promotes apoptosis. Normal cells consequently enter apoptosis at a faster rate than Pol eta-deficient cells. Coincident with hydroxyurea-induced S-phase delay, Pol eta-deficient cells undergo more replication fork breakage and accumulate more foci of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX. We conclude that under conditions of nucleotide deprivation, Pol eta is required for S-phase progression but is proapoptotic. However, as Pol eta is reported to require higher nucleotide concentrations than class B replicative polymerases, its recruitment by hydroxyurea requires it to function under suboptimal conditions. Our results suggest that hydroxyurea-induced apoptosis occurs at the G1/S boundary and that initiation of the S-phase requires greater nucleotide concentrations than does S-phase progression.

Clinical implications of the basic defects in Cockayne syndrome and xeroderma pigmentosum and the DNA lesions responsible for cancer, neurodegeneration and aging.

Cancer, aging, and neurodegeneration are all associated with DNA damage and repair in complex fashions. Aging appears to be a cell and tissue-wide process linked to the insulin-dependent pathway in several DNA repair deficient disorders, especially in mice. Cancer and neurodegeneration appear to have complementary relationships to DNA damage and repair. Cancer arises from surviving cells, or even stem cells, that have down-regulated many pathways, including apoptosis, that regulate genomic stability in a multi-step process. Neurodegeneration however occurs in nondividing neurons in which the persistence of apoptosis in response to reactive oxygen species is, itself, pathological. Questions that remain open concern: sources and chemical nature of naturally occurring DNA damaging agents, especially whether mitochondria are the true source; the target tissues for DNA damage and repair; do the human DNA repair deficient diseases delineate specific pathways of DNA damage relevant to clinical outcomes; if naturally occurring reactive oxygen species are pathological in human repair deficient disease, would anti-oxidants or anti-apoptotic agents be feasible therapeutic agent?

Cockayne syndrome exhibits dysregulation of p21 and other gene products that may be independent of transcription-coupled repair.

Cockayne syndrome (CS) is a progressive childhood neurodegenerative disorder associated with a DNA repair defect caused by mutations in either of two genes, CSA and CSB. These genes are involved in nucleotide excision repair (NER) of DNA damage from ultraviolet (UV) light, other bulky chemical adducts and reactive oxygen in transcriptionally active genes (transcription-coupled repair, TCR). For a long period it has been assumed that the symptoms of CS patients are all due to reduced TCR of endogenous DNA damage in the brain, together with unexplained unique sensitivity of specific neural cells in the cerebellum. Not all the symptoms of CS patients are however easily related to repair deficiencies, so we hypothesize that there are additional pathways relevant to the disease, particularly those that are downstream consequences of a common defect in the E3 ubiquitin ligase associated with the CSA and CSB gene products. We have found that the CSB defect results in altered expression of anti-angiogenic and cell cycle genes and proteins at the level of both gene expression and protein lifetime. We find an over-abundance of p21 due to reduced protein turnover, possibly due to the loss of activity of the CSA/CSB E3 ubiquitylation pathway. Increased levels of p21 can result in growth inhibition, reduced repair from the p21-PCNA interaction, and increased generation of reactive oxygen. Consistent with increased reactive oxygen levels we find that CS-A and -B cells grown under ambient oxygen show increased DNA breakage, as compared with xeroderma pigmentosum cells. Thus the complex symptoms of CS may be due to multiple, independent downstream targets of the E3 ubiquitylation system that results in increased DNA damage, reduced transcription coupled repair, and inhibition of cell cycle progression and growth.

Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Xeroderma pigmentosum (XP) is an autosomal recessive human disease, characterized by an extreme sensitivity to sunlight, caused by the inability of cells to repair UV light-induced damage to DNA. Cell fusion was used to transfer fragments of Chinese hamster ovary (CHO) chromosomes into XP cells. The hybrid cells exhibited UV resistance and DNA repair characteristics comparable to those expressed by CHO cells, and their DNA had greater homology with CHO DNA than did the DNA from XP cells. Control experiments consisted of fusion of irradiated and unirradiated XP cells and repeated exposure of unfused XP cells to UV doses used for hybrid selection. These treatments did not result in an increase in UV resistance, repair capability, or homology with CHO DNA. The hybrid cell lines do not, therefore, appear to be XP revertants. The establishment of these stable hybrid cell lines is an initial step toward identifying and cloning CHO DNA repair genes that complement the XP defect in human cells. The method should also be applicable to cloning genes for other diseases, such as ataxia-telangiectasia and Fanconi's anemia.

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.

Alternative recombination pathways in UV-irradiated XP variant cells.

XP variant (XP-V) cells lack the damage-specific polymerase eta and exhibit prolonged replication arrest after UV irradiation due to impaired bypass of UV photoproducts. To analyse the outcome of the arrested replication forks, homologous recombination (HR, Rad51 events) and fork breakage (Rad50 events) were assayed by immunofluorescent detection of foci-positive cells. Within 1 h of irradiation, XP-V cells showed more Rad51-positive cells than normal cells, while neither cell type showed an increase in Rad50 foci. Beyond 1 h, the frequency of Rad51-positive cells reached similar levels in both cell types, then declined at higher UV doses. At these later times, Rad50-positive cells increased with dose and to a greater extent in XP-V cells. Few cells were simultaneously positive for both sets of foci, suggesting a mutually exclusive recruitment of recombination proteins, or that these pathways operate at different stages during S phase. Analysis of cells containing a vector of tandemly arranged enhanced green fluorescent protein genes also showed that UV-induced HR was higher in XP-V cells. These results suggest that cells make an early commitment to HR, and that at later times a subset of arrested forks degrade into double-strand breaks, two alternative pathways that are greater in XP-V cells.

Increased repair replication in human lymphoid cells by inhibition of polyadenosine diphosphoribose synthesis with no increase in patch sizes.

Repair replication of alkylation damage in WIL-2 lymphoid cells is increased up to 7-fold by addition of 3-aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase. This increase occurs without any change in the repair replication patch size and must therefore represent a large increase in the number of patches. The increase in the number of patches occurs without concomitant increase in the rate of excision of damaged sites. Therefore, it seems unlikely that 3-aminobenzamide plays any role in regulating ligation of repair patches, as commonly supposed. Instead, by inhibiting polyadenosine diphosphoribose polymerase or by other side effects, 3-aminobenzamide appears to elicit random nuclease attack of cellular DNA. The sites of attack are then repaired with patches of similar size, as are most other lesions. Nuclease attack may play a role in the increased cellular toxicity attendant on growth in 3-aminobenzamide.

Relative importance of incision and polymerase activities in determining the distribution of damaged sites that are mended in xeroderma pigmentosum group C cells.

Those pyrimidine dimers that are repaired in confluent xeroderma pigmentosum Group C cells are clustered together in the genome. Although the average level of repair in this complementation group is of the order of 25% of normal, this percentage represents normal levels of repair in one quarter of the genome and little repair in the remainder. The factors that regulate this clustering process have been investigated using inhibitors of the initial incision step of repair (novobiocin) and of the polymerization step (aphidicolin). Novobiocin at a concentration that permitted 30% of repair to continue reduced the clustering of mended sites only slightly. Aphidicolin, in contrast, at a concentration that permitted 30 to 60% of repair to continue caused the mended sites to be distributed randomly. The clustering of repair sites seen in xeroderma pigmentosum Group C cells, therefore, is produced by an excision repair mechanism in which an aphidicolin-sensitive DNA polymerase, presumably alpha, plays an important regulatory role in determining which damaged sites are mended.

Inactivation of ultraviolet repair in normal and xeroderma pigmentosum cells by methyl methanesulfonate.

Excision repair of ultraviolet damage in the DNA of normal and xeroderma pigmentosum (Groups C, D, and variant) cells was inactivated by exposure of cells to methyl methanesulfonate immediately before irradiation independent of the presence of 0 to 10% fetal calf serum. The inactivation could be represented by a semilog relationship between the amount of repair and methyl methanesulfonate concentration up to approximately 5 mM. The inactivation can be considered to occur as the result of alkylation of a large (about 10(6) daltons) repair enzyme complex, and the dose required to reduce repair to 37% for most cells types was between 4 and 7 mM. No consistent, large difference in sensitivity to methyl methanesulfonate was found in any xeroderma pigmentosum complementation group compared to normal cells, implying that reduced repair in these groups may be caused by small inherited changes in the amino acid composition (i.e., point mutations or small deletions) rather than by losses of major components of the repair enzyme complex.

Repair of psoralen-induced cross-links and monoadducts in normal and repair-deficient human fibroblasts.

SV40-transformed normal, xeroderma pigmentosum (XP) and Fanconi's anemia (FA) fibroblasts have distinct repair capacities for monoadducts and DNA interstrand cross-links produced by exposure to near-UV (320-400 nm) light in the presence of 8-methoxypsoralen or angelicin. Excision repair of monoadducts occurred rapidly in normal and FA cells after exposure but not in XP cells. Cross-links were repaired in normal cells with a t1/2 of about 10 h but not in XP or FA cells. When the total number of adducts induced by 8-methoxypsoralen in normal cells was kept constant, the amount of repair replication decreased as the ratio of cross-links to monoadducts increased. This suggests either that cross-link repair is significantly different from monoadduct repair, involving smaller patches and a much slower rate of patching or that cross-links can inhibit monoadduct repair. Our results show that XP group A and FAH12 cell lines are deficient in cross-link repair. The data also suggest that the mechanism of cross-link repair in human cells involves several enzymes and that different ones may be deficient in XP and FA cells.

3-Aminobenzamide can act as a cocarcinogen for ultraviolet light-induced carcinogenesis in mouse skin.

Chronic irradiation (three times a week) with ultraviolet B light of the skin of hairless mouse Uscd (Hr) strains resulted in the induction of skin tumors after 25 to 41 weeks. Topical applications of 3-aminobenzamide (3AB; 0.1 or 1 M) after each irradiation significantly shortened the earliest time of onset of tumors to 13 to 25 weeks and increased the number of animals that developed tumors over 41 weeks from 67% without 3AB to 73% and 81% with 0.1 and 1 M 3AB, respectively. 3-Aminobenzamide has previously been shown to inhibit radiation-induced transformation in vitro. In vivo, 3AB has the opposite effect, indicating the need for caution in extrapolating from in vitro systems to carcinogenesis in vivo.

Absence of specificity in inhibition of DNA repair replication by DNA-binding agents, cocarcinogens, and steroids in human cells.

Although many chemicals, including cocarcinogens, DNA-binding agents, and steroids, inhibit repair replication of ultraviolet-induced damage to DNA in human lymphocytes and proliferating cells in culture, none of these chemicals is specific. Our results show that all the chemicals than they inhibit repair replication. There is thus no cocarcinogens are specific inhibitors of DNA repair or that any of the chemicals studied might be useful adjuncts to tumor therapy merely because of specific inhibition of radiation repair mechanisms.

Effect of 3-aminobenzamide on the rate of ligation during repair of alkylated DNA in human fibroblasts.

3-Aminobenzamide, an inhibitor of polyadenosine diphosphoribose polymerase, produced rapid reversible changes in single-strand break frequencies in DNA from primary human fibroblasts damaged by alkylating agents, but it did not cause such changes in the DNA of cells damaged by ultraviolet light. The increase in single-strand peak frequencies was not due to an accumulation of blocked repair sites, such as occurs with DNA polymerase inhibitors, but to a delay in the rejoining of induced breaks. 3-Aminobenzamide increases the net break frequency that results from a dynamic balance between excision and ligation. This balance appears to be regulated at the ligation step by adenosine diphosphate ribosylation, which is rapidly altered by addition or removal of 3-aminobenzamide. The rapidity with which strand break frequencies change in the presence of 3-aminobenzamide implies that individual strand breaks resulting from excision at any time after exposure have a lifetime of no more than about 30 min in the cell.

Induction of DNA-DNA cross-link formation in human cells by various psoralen derivatives.

Furocoumarin-induced DNA damage, monoadducts, and cross-links were measured in normal human, xeroderma pigmentosum, and Fanconi's anemia cells after exposure to near-UV (356 nm). At similar concentrations and near-UV doses, photoaddition by 8-methoxypsoralen was twice that by angelicin and the substitution of bromodeoxyuridine for thymidine in one strand of DNA did not alter the binding. The rate of cross-linking by 8-methoxypsoralen was twice that of 5-methoxypsoralen. Low frequencies of cross-links were detected from angelicin and 3-carbethoxypsoralen but none were detected from 5-geranoxypsoralen at concentrations up to 25 micrograms/ml and near-UV doses up to 45,000 J/m2.

Repair and replication of DNA in hereditary (bilateral) retinoblastoma cells after X-irradiation.

Fibroblasts from patients with hereditary retinoblastoma reportedly exhibit increased sensitivity to killing by X-rays. Although some human syndromes with similar or greater hypersensitivity to DNA-damaging agents (e.g., X-rays, ultraviolet light, and chemical carcinogens), such as xeroderma pigmentosum, are deficient in DNA repair, most do not have such clearly demonstrable defects in repair. Retinoblastoma cells appear to be normal in repairing single-strand breaks and performing repair replication after X-irradiation and also in synthesizing poly(adenosine diphosphoribose). Semiconservative DNA replication in these cells, however, is slightly more resistant than normal after X-irradiation, suggesting that continued replication of damaged parental DNA could contribute to the pathogenesis of the disease. This effect is small, however, and may be a consequence rather than a cause of the fundamental enzymatic abnormality in retinoblastoma that causes the tumorigenesis.