James E. Cleaver

CELL SURVIVAL CHARACTERISTICS AND MOLECULAR RESPONSES OF ANTARCTIC PHYTOPLANKTON TO ULTRAVIOLET‐B RADIATION

Twelve species of Antarctic diatoms were studied to assess UV sensitivity in relation to cellular and molecular aspects of DNA damage and repair. Responses of cell survival, induction of DNA damage, and DNA repair capacity were determined. There was a wide range of interspecific UV‐sensitivity among diatoms. D37 values (average fluence to kill one cell) ranged from 681 J · m−2 (most sensitive) to 25,338 J · m−2 (most resistant). Molecular analysis (by radioimmunoassay) of UV‐induced DNA damage [induction of cys‐syn cyclobutane dimers and pyrimidine (6‐4) pyrimidone photoproducts] also revealed considerable variability among species [0.98–84 lesions · (108 daltons DNA)−1 induced by exposure to 2500 J · m−2]. Repair of DNA damage ranged from 0.18 to 2.72 lesions removed · (108 daltons DNA)−1 in 6 h; removal represented 0.72–73.5% of initial damage. Comparison of cellular responses associated with photoenhanced repair and nucleotide excision (“dark”) repair indicated that light‐mediated correction of UV damage was an important factor in cell survival. There was a relationship between the number of photoproducts induced and cell survival, but not between repair efficiency and survival. The data also indicate a general dependence of photoproduct induction and D37 values on cell size and shape (expressed as the surface area: volume ratio which ranged from 0.07 to 0.66 between species) and suggest that these factors are indicators of UV sensitivity. Smaller cells with greater surface area: volume ratios sustained more damage per unit of DNA, had lower D37 values, and were more sensitive to UV exposure. The wide species variations observed in molecular and cellular responses to UV exposure emphasize the ecological implications of changes in natural UV regimes. These changes can act as determinants of cell size and taxonomic structure within phytoplankton communities and have as yet unknown effects on trophic interactions within the Antarctic ecosystem.

Cancer in xeroderma pigmentosum and related disorders of DNA repair

Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins.

Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity

Mutations in genes on the nucleotide excision repair pathway are associated with diseases, such as xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that involve skin cancer and developmental and neurological symptoms. These mutations cause the defective repair of damaged DNA and increased transcription arrest but, except for skin cancer, the links between repair and disease have not been obvious. Widely different clinical syndromes seem to result from mutations in the same gene, even when the mutations result in complete loss of function. The mapping of mutations in recently solved protein structures has begun to clarify the links between the molecular defects and phenotypes, but the identification of additional sources of clinical variability is still necessary.

Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ∼75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

A screening method for isolating DNA repair-deficient mutants of CHO cells.

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV), ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D37 dose of 1.0 J/m2 compared to 7.0 J/m2 for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m2. Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of60Co γ-rays.

A summary of mutations in the UV-sensitive disorders: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

The human diseases xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy are caused by mutations in a set of interacting gene products, which carry out the process of nucleotide excision repair. The majority of the genes have now been cloned and many mutations in the genes identified. The relationships between the distribution of mutations in the genes and the clinical presentations can be used for diagnosis and for understanding the functions and the modes of interaction among the gene products. The summary presented here represents currently known mutations that can be used as the basis for future studies of the structure, function, and biochemical properties of the proteins involved in this set of complex disorders, and may allow determination of the critical sites for mutations leading to different clinical manifestations. The summary indicates where more data are needed for some complementation groups that have few reported mutations, and for the groups for which the gene(s) are not yet cloned. These include the Xeroderma pigmentosum (XP) variant, the trichothiodystrophy group A (TTDA), and ultraviolet sensitive syndrome (UVs) groups. We also recommend that the XP‐group E should be defined explicitly through molecular terms, because assignment by complementation in culture has been difficult. XP‐E by this definition contains only those cell lines and patients that have mutations in the small subunit, DDB2, of a damage‐specific DNA binding protein. Hum Mutat 14:9–22, 1999.

UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization

UV-induced replication arrest in the xeroderma pigmentosum variant (XPV) but not in normal cells leads to an accumulation of the Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX (γ-H2AX) in large nuclear foci at sites of stalled replication forks. These complexes have been shown to signal the presence of DNA damage, in particular, double-strand breaks (DSBs). This finding suggests that UV damage leads to the formation of DSBs during the course of replication arrest. After UV irradiation, XPV cells showed a fluence-dependent increase in the yield of γ-H2AX foci that paralleled the production of Mre11 foci. The percentage of foci-positive cells increased rapidly (10–15%) up to fluences of 10 J⋅m−2 before saturating at higher fluences. Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h after UV irradiation. This pattern contrasts sharply to the situation observed after x-irradiation, where peak levels of γ-H2AX foci were found to precede the formation of Mre11 foci by several hours. The nuclear distributions of γ-H2AX and Mre11 were found to colocalize spatially after UV- but not x-irradiation. UV-irradiated XPV cells showed a one-to-one correspondence between Mre11 and γ-H2AX foci-positive cells. These results show that XPV cells develop DNA DSBs during the course of UV-induced replication arrest. These UV-induced foci occur in cells that are unable to carry out efficient bypass replication of UV damage and may contribute to further genetic variation.

It was a very good year for DNA repair

James E. Cleaver Laboratory of Radiobiology and Environmental Health University of California San Francisco, California 94143-0750 The proposal of a mutator phenotype, which develops as an early step in the formation of human tumors and results in an intrinsic genetic instability (Loeb, 1991), received support experimentally with the discovery of unstable mi- crosatellite repeat sequences in sporadic colon tumors and in hereditary nonpolyposis colon cancer (HNPCC) (Aaltonen et al., 1993). A majority of these developing tu- mors contain frequent deletions and insertions within (CA), and other simple repeated sequences. This increased fre- quency of mutations within the repeated sequences of an HNPCC cell line was characterized as a replication error (RER+) phenotype that presumably develops early in the progression of the tumor (Parsons et al., 1993). A genetic instability that produces an expansion of trinucleotide re- peats has also been observed in several hereditary neuro- logical and neuromuscular diseases, including Hunting- ton’ s disease and fragile X syndrome (Martin, 1993). The mechanism underlying this trinucleotide expansion is un- known. For the HNPCC tumors, however, evidence is emerging that adefect in DNA repair is a major contributor to the observed genetic instability. The cloning of a human mismatch repair gene,

Temporal Dissection of Tumorigenesis in Primary Cancers

Timely intervention for cancer requires knowledge of its earliest genetic aberrations. Sequencing of tumors and their metastases reveals numerous abnormalities occurring late in progression. A means to temporally order aberrations in a single cancer, rather than inferring them from serially acquired samples, would define changes preceding even clinically evident disease. We integrate DNA sequence and copy number information to reconstruct the order of abnormalities as individual tumors evolve for 2 separate cancer types. We detect vast, unreported expansion of simple mutations sharply demarcated by recombinative loss of the second copy of TP53 in cutaneous squamous cell carcinomas (cSCC) and serous ovarian adenocarcinomas, in the former surpassing 50 mutations per megabase. In cSCCs, we also report diverse secondary mutations in known and novel oncogenic pathways, illustrating how such expanded mutagenesis directly promotes malignant progression. These results reframe paradigms in which TP53 mutation is required later, to bypass senescence induced by driver oncogenes.

Absence of excision of ultraviolet-induced cyclobutane dimers in xeroderma pigmentosum.

XERODERMA pigmentosum is a rare human skin disease which is inherited as an autosomal recessive mutation and has been known by dermatologists for nearly a century [ 1-31. The major clinical symptom is high actinic skin carcinogenesis [ 1.4.51. There are two forms of the disease: one shows only the skin symptoms, the other more uncommon form shows additional neurological symptoms and is known as the de Sanctis Cacchione syndrome [5-71. Whether the neurological symptoms should be regarded as part of xeroderma pigmentosum or due to separate genetic factors is as yet unknown [2,5-71. Fibroblasts[& 91 and epithelial cells[lO] from the skin in both forms of this disease appear to perform low or negligible amounts of DNA repair replication after irradiation with ultraviolet (u.v.) light, but the level of such residual amounts cannot yet be correlated with the two forms of the disease since the number of cases studied thus far is too small[& 111. Previous studies on DNA repair in de Sanctis Cacchione skin fibroblasts suggested that an early step in DNA repair involving enzymatic strand breakage is defective [9,12]. Such an hypothesis has also been advanced by Setlow et al. [ 131 for skin fibroblasts from the non-neurological form of xeroderma pigmentosum. They showed that thymine-containing dimers are excised after U.V. damage of normals cells but not of xeroderma cells. Since the two forms of the disease might show some biochemical differences, we have now investigated the excision of thyminecontaining dimers in cultured cells from patients with the de Sanctis Cacchione syndrome. Preliminary results of this investigation have already been reported [9, 121. Sterile 1 mm punch biopsies were taken from apparently normal regions of the arms of three de Sanctis Cacchione patients and two normal persons. Fibroblast cultures were developed in Eagles minimum essential medium with 3 mglml dextrose, non-essential amino acids, and 15 percent fetal calf serum. Usually, 1-5-2 months elapsed between the time of biopsy and the start of any experiments with fibroblast cultures. They were then used throughout the period of steady growth until cultures degenerated[ 14, IS]. Human embryonic fibroblasts (WI-38) were also used and these