Development of a Greenhouse Screening Method for Adult Plant Response in Wheat to Stem Rust.

Screening for adult plant resistance in wheat to stem rust, caused by Puccinia graminis f. sp. tritici, is generally conducted in field plots. Although such evaluations are successful if managed properly, field ratings are time consuming, expensive, weather dependent, and open to inoculum of unwanted races or other confounding diseases. The objective of this study was to develop a dependable system of screening the response of adult plants to stem rust under greenhouse conditions. A comparison of inoculation methods and incubation environments showed that plants inoculated with urediniospores suspended in water, followed by a 24 h dew period in a plastic chamber constructed in a greenhouse, gave the most consistent results. Measurements of response type, stem rust severity, and frequency in follow-up experiments indicated that the most reliable infection was obtained when plants sprayed with 1.25 mg urediniospores per ml water were incubated in the plastic chamber. Using the optimized protocol, a Kariega × Avocet S doubled haploid population was inoculated with two P. graminis f. sp. tritici races. Depending on the race, composite interval mapping showed flag leaf infection type to be significantly influenced by regions on chromosomes 6A, 6D, and 7D. Stem rust severity and reaction type mapped to chromosomes 6D and/or 6A. The Lr34/Yr18/Sr57 gene derived from Kariega on chromosome 7D affected the rust response on flag leaves but not on stems of greenhouse-grown plants. This study showed that phenotyping and genetic analysis of especially major effect stem rust resistance in adult wheat plants is possible and reproducible under controlled conditions in a greenhouse.

Loss-induced suppression and revival of lasing.

Controlling and reversing the effects of loss are major challenges in optical systems. For lasers, losses need to be overcome by a sufficient amount of gain to reach the lasing threshold. In this work, we show how to turn losses into gain by steering the parameters of a system to the vicinity of an exceptional point (EP), which occurs when the eigenvalues and the corresponding eigenstates of a system coalesce. In our system of coupled microresonators, EPs are manifested as the loss-induced suppression and revival of lasing. Below a critical value, adding loss annihilates an existing Raman laser. Beyond this critical threshold, lasing recovers despite the increasing loss, in stark contrast to what would be expected from conventional laser theory. Our results exemplify the counterintuitive features of EPs and present an innovative method for reversing the effect of loss.

Identification of adult plant resistance to stripe rust in the wheat cultivar Cappelle-Desprez.

Following the appearance of stripe rust in South Africa in 1996, efforts have been made to identify new sources of durable resistance. The French cultivar Cappelle-Desprez has long been considered a source of durable, adult plant resistance (APR) to stripe rust. As Cappelle-Desprez contains the seedling resistance genes Yr3a and Yr4a, wheat lines were developed from which Yr3a and Yr4a had been removed, while selecting for Cappelle-Desprez derived APR effective against South African pathotypes of the stripe rust fungus, Puccinia striiformis f. sp. tritici. Line Yr16DH70, adapted to South African wheat growing conditions, was selected and crossed to the stripe rust susceptible cultivar Palmiet to develop a segregating recombinant inbred line mapping population. A major effect QTL, QYr.ufs-2A was identified on the short arm of chromosome 2A derived from Cappelle-Desprez, along with three QTL of smaller effect, QYr.ufs-2D, QYr.ufs-5B and QYr.ufs-6D. QYr.ufs-2D was located within a region on the short arm of chromosome 2D believed to be the location of the stripe rust resistance gene Yr16. An additional minor effect QTL, QYr.ufs-4B, was identified in the cv. Palmiet. An examination of individual RILs carrying single or combinations of each QTL indicated significant resistance effects when QYr.ufs-2A was combined with the three minor QTL from Cappelle-Desprez, and between QYr.ufs-2D and QYr.ufs-5B.

First Report of a New Wheat Leaf Rust (Puccinia triticina) Race with Virulence for Lr12, 13, and 37 in South Africa.

A new race of Puccinia triticina was collected from common wheat (Triticum aestivum) in the Eastern and Western Cape provinces during the annual rust survey in 2009. Six single-pustule isolates from a field collection, which were shown to be a new race in preliminary analyses, were inoculated onto seedlings of 16 Thatcher (Tc) near-isogenic differential lines (1) and other tester lines with known Lr genes. Standard procedures for inoculation, incubation, and rust evaluation were followed (4) and all infection studies were repeated. The low infection type of Lr18 was confirmed at 18°C. All six isolates were avirulent (infection types [ITs] 0; to 2) to Lr1, 2a, 2c, 9, 11, 16, 18, and 24 and virulent (ITs 3 to 4) to Lr3, 3ka, 10, 14a, 17, 26, 30, B, and Tc (control). The new race, named 3SA145 according to the ARC-Small Grain Institute notation, corresponds to race CCPS in the North American system (1). On the basis of seedling ITs of the extended Lr gene set, 3SA145 was avirulent (ITs 0; to 22+) to Lr2b, 19, 21, 23, 25, 28, 29, 32, 36 (E84081), 38, 45, 47 (KS90H450), 50 (KS96WGRC36), 51 (R05), and 52 and virulent to Lr3bg, 15, 20 (Thew), 27+31 (Gatcher), and 33. Lines containing the adult plant resistance (APR) genes Lr12 (RL6011, IT 3++), Lr13 (CT263, IT 3), Lr22b (Tc, IT 4), and Lr37 (RL6081, IT 3) were susceptible in the adult stage to 3SA145, whereas lines with the APR genes Lr22a (RL6044, IT ;1), Lr34 (RL6058, IT Z1), and Lr35 (RL6082, IT ;1) were resistant in controlled infection studies in a greenhouse. A control, the common race (3SA133), was virulent only on Tc adult plants. In seedlings, 3SA133 was avirulent to Lr15, 17, 26, and 27+31, but unlike 3SA145, it was virulent to Lr1, 2c, 11, 18, 24, and 28. Races 3SA133 and 3SA145 did not differ in their virulence to the remaining seedling genes. Virulence to Lr37 has been reported in several countries, including Australia, Canada, Uruguay, and the United States (1,2). Prior to the detection of 3SA145, adult plants of RL6081 were resistant to all wheat leaf rust races in South Africa. In 2009, however, RL6081 showed severity levels of up to 30S at certain Western Cape trap plot sites. Of 124 South African bread wheat cultivars and advanced breeding lines tested at the seedling stage, 3SA145 was virulent to 48, whereas 3SA133 was virulent to 36 entries. A further six entries were heterogeneous in their reaction to 3SA145. In adult plant infection studies of 48 South African spring wheats in a greenhouse, 19 were susceptible (flag leaf IT ≥3) and 22 were resistant to 3SA145. Seven entries showed a Z3 flag leaf IT indicating adult plant resistance. According to a simple sequence repeat (SSR) study using 17 primer-pair combinations described by Szabo and Kolmer (3), 3SA145 showed 30% homology with the dominant South African races. Although virulence to Lr12 and Lr13 has been known in different leaf rust races in South Africa, to our knowledge, this is the first report of combined virulence to Lr12, 13, and 37. The SSR data and unique avirulence/virulence profile suggest that 3SA145 may be an exotic introduction to South Africa. References: (1) J. A. Kolmer et al. Plant Dis. 89:1201, 2005. (2) B. McCallum and P. Seto-Goh. Can. J. Plant Pathol. 31:80, 2009. (3) L. Szabo and J. Kolmer. Mol. Ecol. Notes 7:708, 2007. (4) T. Terefe et al. S. Afr. J. Plant Soil 26:51, 2009.

First Report of Virulence for the Wheat Leaf Rust (Puccinia triticina) Resistance Gene Lr32 in South Africa.

The wheat leaf rust resistance gene Lr32 was transferred from Aegilops tauschii Coss. to bread wheat (Triticum aestivum L.) (1). Despite virulence for Lr32 in some isolates from Bulgaria, Israel, and Turkey, the gene has been reported to be effective in Australia, Mexico, the United States, and South Africa (1,2). A leaf rust isolate that differed in its avirulence/virulence profile from previously recorded races of Puccinia triticina Eriks. in South Africa was collected from triticale (× Triticosecale) in the Western Cape in 2005. According to the South African leaf rust differential set (3), this isolate (UVPt19) was avirulent for Lr3a, 3bg, 3ka, 10, 11, 16, 20, 26, and 30 and virulent for Lr1, 2a, 2b, 2c, 14a, 15, 17, 24, and Thatcher (Tc, control). Except for Lr20 in cv. Thew, all differentials are Tc near-isogenic lines. In comparison with known South African races (3), it differed from race 3SA132 at the Lr10 locus. Using standard rust pathology protocols (3), an expanded set of Lr gene lines (non Tc lines indicated) showed that UVPt19 is avirulent on wheat seedlings containing Lr9, 19, 21, 25, 27+31 (Gatcher), 29, 36 (ER84018), 37, 41 (KS91WGRC10), 44, 45, 47 (KS90H450), 50 (KS96WGRC36), 51 (R05), and 52, and virulent for Lr12, 22a, 23, 28, 32, 33, and 35. In the seedling stage, UVPt19 was virulent for the temperature sensitive genes Lr13, 18, and 34 at 25°C, but produced lower infection types (ITs) on Lr18 and 34 at 14 to 18°C. Seedlings of Pavon 76 (Lr46) were resistant (IT ;1=) to UVPt19. The susceptible response of lines carrying Lr32 was confirmed by high ITs (3++4) on RL5713/2*Mq, RL6086 (TcLr32), and RL5713/2*Mq//6*Palmiet. A control isolate (UVPt9) produced ITs ;1+, ;1+, and ;;1= on these lines, respectively. UVPt19 was virulent on line RL6092 (TcLr20) but avirulent on Thew. When tested on adult plants of lines RL6011 (TcLr12), CT263 (TcLr13), RL6044 (TcLr22a), RL6058 (TcLr34), RL6082 (TcLr35), RL6081 (TcLr37), and Tc (control), UVPt19 was only virulent (IT 3+) on CT263 and Tc. Flag leaves of RL6011 (IT ;1), RL6044 (IT 1), RL6058 (IT Z3-), RL6082 (IT 0;), and RL6081 (IT ;1) were resistant. UVPt19 was virulent on seedlings of 11 of 13 triticale cultivars and lines tested as opposed to UVPt9, which was virulent to only one entry. From a collection of 105 South African bread wheat cultivars and elite breeding lines, UVPt19 was virulent on 13 and five were mixed in their response to this isolate. All IT experiments were repeated. Although virulence has emerged for Lr32 in South Africa, the gene has not been used in local cultivars. Previously, McIntosh et al. (1) also reported that Lr32 has not been exploited in wheat production. On the basis of current evidence, UVPt19 appears to be potentially more damaging to triticale than bread wheat. Furthermore, the race seems rare because it was not collected in a recent wheat leaf rust survey in South Africa (3). References: (1) R. A. McIntosh et al. The Wheat Rusts: An Atlas of Resistance Genes, CSIRO-Kluwer, Dordrecht, the Netherlands, 1995. (2) Z. A. Pretorius. Phytophylactica 21:195, 1989. (3) T. Tarekegn et al. S. Afr. J. Plant Soil 26:51, 2009.

First Report of a Puccinia graminis f. sp. tritici Race Virulent to the Sr24 and Sr31 Wheat Stem Rust Resistance Genes in South Africa.

Isolates of Puccinia graminis f. sp. tritici belonging to the Ug99 race group are virulent to a broad spectrum of resistance genes, rendering most of the world's wheat germplasm susceptible to stem rust (3). Following the initial detection of Ug99 (TTKSK, North American [NA] race notation) in Uganda, virulence to the widely used Sr31 resistance gene has been reported from Kenya, Ethiopia, Sudan, and Iran (2,3). In November 2009, a wheat genotype suspected to carry Sr31 showed a susceptible response to stem rust in a disease nursery (29°08'05.02''S, 30°38'29.18''E), inoculated with race TTKSP, near Greytown in KwaZulu-Natal, South Africa. Inoculation of urediniospores of the field collection (isolate UVPgt60) onto seedlings of line Federation4*/Kavkaz confirmed virulence for Sr31. In three independent, replicated, and comparative seedling tests, eight single-pustule isolates of UVPgt60 all typed to race PTKST following the NA race nomenclature. These isolates produced compatible infection types (ITs) (3+ to 4) on the Sr31 testers Gamtoos, Sr31/6*LMPG, Federation4*/Kavkaz, Kavkaz, and Clement, whereas isolate UVPgt59 (TTKSP) was avirulent (ITs ;1 to 1) on these genotypes. In addition to Sr31 virulence, the new race differed from TTKSP by producing a lower IT (2 to 2++) on Cns_T.mono_ deriv., the accepted entry for Sr21 in the NA differential set. The UVPgt60 isolates were clearly avirulent on Einkorn (Sr21) (IT ;1=), a response that also differed from those produced by BPGSC, TTKSF, and TTKSP (IT 2). With the exception of Sr21, UVPgt60 isolates had a virulence pattern similar to race TTKST (1), notably the virulence combination for Sr24 and Sr31. Isolate UVPgt60.6 was randomly selected for testing on additional Sr genes and South African wheat cultivars and breeding lines. Similar to the race identification experiments seedling tests were duplicated and compared with reactions produced by TTKSP and other races. Greenhouse temperatures for all seedling tests ranged between 18 and 25°C. On the basis of primary leaf responses, PTKST is avirulent (ITs 0; to 2++) for Sr13, 14, 21, 22, 25, 26, 27, 29, 32, 33, 35, 36, 37, 39, 42, 43, 44, Em, Tmp, and Satu and virulent (ITs 3 to 4) for Sr5, 6, 7b, 8a, 8b, 9a, 9b, 9d, 9e, 9g, 10, 11, 16, 17, 24, 30, 31, 34, 38, 41, and McN. From 103 South African wheat cultivars and lines tested as seedlings, 59 and 47 were susceptible (IT ≥ 3) to races PTKST and TTKSP, respectively. Simple-sequence repeat analysis (4) with selected primer pairs showed that PTKST clusters with isolates belonging to the Ug99 lineage. Subsequent to the collection made at Greytown, stem rust sampled in December 2009 from naturally infected breeders' lines at Cedara (29°32'19.59''S, 30°16'03.50''E), KwaZulu-Natal, revealed five isolates with a virulence profile similar to PTKST. On the basis of current evidence it appears that PTKST may be an introduction to South Africa rather than a single-step mutation from local stem rust races. References: (1) Y. Jin et al. Plant Dis. 92:923, 2008. (2) K. Nazari et al. Plant Dis. 93:317, 2009. (3) R. P. Singh et al. Adv. Agron. 98:271, 2008. (4) B. Visser et al. Mol. Plant Pathol. 10:213, 2009.

Mismatch repair deficiency and CpG island hypermethylation in sporadic colon adenocarcinomas.

Many studies have documented CpG island hypermethylation in human colon adenocarcinomas. Several of these reports have additionally found such CpG island hypermethylation to be more extensive in tumors with a mismatch-repair deficiency, as revealed by microsatellite instability (MSI+). Because the source of samples used in these prior studies may not have been representative of the general population, we have reinvestigated this issue using samples from a population-based study. A total of 15 MSI+ tumors were identified, and they were compared with 47 MSI- tumors that were similar in distribution by age, sex, and race. Microdissected tumor and normal adjacent mucosal DNA samples from each patient were subjected to a quantitative DNA methylation analysis at 13 separate CpG dinucleotides located in five CpG islands in four different genes [APC, ESR1 (ER), CDKN2A (p16; promoter and exon 2), and MLH1]. Four of five CpG islands showed a statistically significantly increased level of methylation in tumor tissue compared with adjacent normal mucosa. In contrast to previous studies, we did not find any statistically significant correlations between MSI status and methylation levels of any of the CpG islands other than MLH1. Furthermore, we observed a positive correlation between MLH1 methylation and CDKN2A methylation (P = 0.03), whereas no association was noted between MSI positivity and CDKN2A methylation (P = 0.95). The latter results suggest a possible defect in the protection against CpG island hypermethylation shared between CDKN2A and MLH1 and do not support the notion of a functional association between CDKN2A methylation and the phenotype of mismatch repair deficiency.

Cognitive function and reproductive hormones in adjuvant therapy for breast cancer: a critical review.

Deterioration in cognitive function-particularly learning, memory, and attention-has been reported by women with breast cancer who receive adjuvant chemotherapy. Deficits in cognitive function reported by women with breast cancer are similar to those experienced by women as a consequence of natural or surgical menopause. The basis of these deteriorations may include reductions in reproductive hormone levels, particularly estrogens and progesterones, that occur as a result of adjuvant chemotherapy. This paper critically examines the literature related to the impact of adjuvant chemotherapy and reproductive hormone changes on cognitive function in women with breast cancer and suggests direction for future research in this area. The paper proposes a framework for investigation of the problem and discusses the challenges associated with the conduct of this research.

PAX6 methylation and ectopic expression in human tumor cells.

The mechanisms underlying the de novo methylation of CpG islands in human cancer remain almost completely unknown. We used a methylation-sensitive arbitrarily primed polymerase chain reaction (Ms AP-PCR) technique to scan genomic DNA for differential methylation patterns and identified a 550 bp band that was hypermethylated in a majority of colon and bladder cancer cells. This band corresponded to a CpG-rich region in exon 5 of PAX6, which is a highly conserved transcription regulatory factor involved in embryogenesis. Interestingly, exon 5 was very frequently methylated in solid tumors compared with adjacent normal tissues, 17 out of 27 (63%) in bladder, and colon, but the promoter remained unmethylated in all these cases. This methylation was not effective in blocking transcription since ectopic expression of PAX6 was seen in several tumors and cell lines with extensive exon 5 methylation. De novo methylation of the promoter was only seen in tumor cell lines and was associated with gene silencing since treatment with 5-aza-2'-deoxycytidine restored expression to the cells and resulted in a less methylated promoter. Thus, ectopic expression and hypermethylation of exon 5 of PAX6 demonstrate that methylation within a transcribed region, as opposed to promoter methylation, does not block gene expression.

Cognitive function and quality of life in interferon therapy for melanoma.

The purpose of this pilot study was to describe short- and long-term changes in cognitive function and quality of life in patients with melanoma receiving interferon (IFN) alpha-2b. This study used a three-group, repeated measures design in which cognitive function and quality of life were evaluated prior to initiation of treatment at 3-month intervals during treatment and 3 months following the completion of treatment. The sample consisted of 16 adults with Stage II or III melanoma, randomized to one of three treatment groups. Participants in Arm A received high-dose IFN alpha-2b, those in Arm B received low-dose IFN alpha-2b, and those in Arm C received no therapy (control). No significant changes in cognitive function were detected. In participants in Arm A, there was a significant deterioration in the physical well-being dimension of quality of life from baseline to 3 months after beginning therapy.

Roles of cell division and gene transcription in the methylation of CpG islands.

De novo methylation of CpG islands within the promoters of eukaryotic genes is often associated with their transcriptional repression, yet the methylation of CpG islands located downstream of promoters does not block transcription. We investigated the kinetics of mRNA induction, demethylation, and remethylation of the p16 promoter and second-exon CpG islands in T24 cells after 5-aza-2'-deoxycytidine (5-Aza-CdR) treatment to explore the relationship between CpG island methylation and gene transcription. The rates of remethylation of both CpG islands were associated with time but not with the rate of cell division, and remethylation of the p16 exon 2 CpG island occurred at a higher rate than that of the p16 promoter. We also examined the relationship between the remethylation of coding sequence CpG islands and gene transcription. The kinetics of remethylation of the p16 exon 2, PAX-6 exon 5, c-ABL exon 11, and MYF-3 exon 3 loci were examined following 5-Aza-CdR treatment because these genes contain exonic CpG islands which are hypermethylated in T24 cells. Remethylation occurred most rapidly in the p16, PAX-6, and c-ABL genes, shown to be transcribed prior to drug treatment. These regions also exhibited higher levels of remethylation in single-cell clones and subclones derived from 5-Aza-CdR-treated T24 cells. Our data suggest that de novo methylation is not restricted to the S phase of the cell cycle and that transcription through CpG islands does not inhibit their remethylation.

DNA methylation as a target for drug design.

DNA methylation is essential for normal embryonic development. Distinctive genomic methylation patterns must be formed and maintained with high fidelity to ensure the inactivities of specific promoters during development. The mutagenic and epigenetic aspects of DNA methylation are especially interesting because they may lead to the inactivation of genes which are involved in human carcinogenesis. The mutagenicity of 5-Methylcytosine (5mC) and the role of promoter hypermethylation in gene silencing, particularly in cancer, suggest a clinical significance for the design of novel DNA methylation inhibitors which may be utilized to reverse the effects of DNA methylation.

The role of DNA methylation in expression of the p19/p16 locus in human bladder cancer cell lines.

Methylation of CpG sites in the control regions of tumor suppressor genes may be an important mechanism for their heritable, yet reversible, transcriptional inactivation. These changes in methylation may impair the proper expression and/or function of cell cycle regulatory genes and confer a selective growth advantage to affected cells. Detailed methylation analysis using genomic bisulfite sequencing was performed on a series of subclones of a bladder cancer cell line in which a hypermethylated p16 gene had been reactivated by transient treatment with 5-aza-2'-deoxycytidine. Methylation of the CpG island in the promoter of the p16 gene in human bladder cancer cells did not stop the formation of a transcript initiated 20 kb upstream by the p19 promoter but did prevent the expression of a p16 transcript. Furthermore, we show that reactivant clones that expressed p16 at varying levels contained heterogeneous methylation patterns, suggesting that p16 expression can occur even in the presence of a relatively heavily methylated coding region. We also present the first functional evidence that methylation of only a small number of CpG sites can significantly down-regulate p16 promoter activity, thus providing support for the model of progressive inactivation of this tumor suppressor gene by DNA methylation.

Inhibition of DNA methylation by 5-aza-2'-deoxycytidine suppresses the growth of human tumor cell lines.

Alterations in DNA methylation patterns accompany the establishment of immortal cell lines. De novo methylation of CpG islands within the control regions of growth-regulatory genes may inactivate their transcription, giving cells selective growth advantages in culture. We exposed seven human tumor cell lines and two human fibroblast cell strains to the demethylating agent, 5-aza-2'-deoxycytidine (5-Aza-CdR), to determine whether the silencing of growth-regulatory genes by de novo methylation in immortalized cell lines could be reversed, possibly restoring growth control. After recovery from the immediate cytotoxic effects of 5-Aza-CdR, this agent suppressed cellular growth in all seven tumor lines but not in either fibroblast strain. Because alterations in the p16 (CDKN2/MTS1) cell cycle regulatory gene are associated with numerous cancers, we analyzed expression of this gene before and after 5-Aza-CdR treatment. The gene was reactivated by 5-Aza-CdR treatment in three of four tumor cell lines not expressing p16, whereas the fourth tumor line contained a p16 homozygous deletion. p16 was shown to be hypermethylated only in the cell lines and its up-regulation by 5-Aza-CdR was associated with demethylation of the p16 promoter. The remaining tumor lines expressed p16 at constant levels before and after 5-Aza-CdR treatment and showed minimal p16 promoter methylation, suggesting that other growth-regulatory genes may have been silenced by de novo methylation in these cells. p16 expression, cell growth inhibition, and G1 cell cycle arrest by 5-Aza-CdR in the T24 bladder tumor cell line were also heritable after prolonged passage in culture. Furthermore, a dormant p16 gene was reactivated in T24 cells growing in nu/nu rats, and 5-Aza-CdR treatment of T24 cells before inoculation into nu/nu mice decreased the rate of tumor growth. These results suggest that 5-Aza-CdR may slow the growth of tumor cells by reactivating growth-regulatory genes silenced by de novo methylation.

Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

An introduction to oxygen free radicals.

Oxygen free radicals are byproducts from the fundamental metabolic activities within the body. Normally, radicals are neutralized by enzymatic activity or natural antioxidants. Thus the generation of free radicals poses no problem so long as the balance between oxygen radical production and eradication remains in balance. There are multiple medical conditions, such as myocardial infarction, carcinogenesis, and neurologic trauma, to name a few, that may be aggravated by the presence of oxygen free radicals. This article will present an overview of oxygen free radicals: their normal formation and control and how they might further injure tissue in particular diseases. The implications for health care professionals are highlighted.

Potential mechanisms of interferon neurotoxicity.

This article examines potential mechanisms underlying the neurologic toxicities associated with interferon therapy in patients with cancer. The following mechanisms for interferon-related toxicities have been proposed: changes in certain neuroendocrine hormone levels; structural similarities and common pathways between interferon and specific neuroendocrine hormones; and the immunoregulatory effects of interferon. Strategies for the assessment and management of interferon-related neurologic toxicities are discussed. A reduction in dose or discontinuation of interferon may be recommended if interferon neurotoxicity develops. Additional interventions may include reorientation strategies, provision of uninterrupted periods of rest, and safety precautions. Directions for future research are recommended.

Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing.

Loss of heterozygosity on 9p21, where the p16/CDKN2 tumor suppressor and the p15INK4B cell cycle regulator genes are located, is a common genetic alteration in bladder cancer. However, it has been difficult to demonstrate homozygous deletions and intragenic mutations in either of these two genes in primary transitional cell carcinomas (TCC) of the bladder. Similarly, colon cancer-derived cell lines have shown no homozygous deletions of the p16/CDKN2 locus in contrast to a wide variety of tumor-derived cell lines. We have investigated abnormal methylation of the 5' CpG islands of the p16/CDKN2 and p15INK4B genes as an alternative mechanism of inactivation of these genes in bladder and colon cancers. De novo methylation of the 5' CpG island of p16/CDKN2 was observed in 12 of 18 (67%) uncultured bladder TCCs and in 2 of 3 (67%) bladder cell lines. In contrast, only 1 of 10 (10%) colon carcinomas showed methylation of the 5' CpG island of p16/CDKN2. It was striking to find that this region was extensively methylated and the gene not expressed in the normal colonic mucosa of 6 of 10 (60%) patients with colon cancer, whereas 5 of the corresponding colon tumors showed no methylation and high levels of p16/CDKN2 expression. Our data show a significant correlation (P = 0.00001, two-sided) between the absence of p16/CDKN2 expression and methylation of its 5' CpG island in bladder tumors, cell lines, and normal colon mucosa. In contrast, no association was observed between expression and methylation status of the 5' CpG island of p15INK4B. Our results suggest that the p16/CDKN2 tumor suppressor gene may be inactivated by methylation of its 5' CpG island in TCCs of the bladder. We also present evidence of methylation of the 5' CpG island in this autosomal gene in normal colonic tissue.