Molecular Markers Help with Breeding for Agronomic Traits of Spring Wheat in Kazakhstan and Siberia.

The Kazakhstan-Siberia Network for Spring Wheat Improvement (KASIB) was established in 2000, forming a collaboration between breeding and research programs through biannual yield trials. A core set of 142 genotypes from 15 breeding programs was selected, genotyped for 81 DNA functional markers and phenotyped for 10 agronomic traits at three sites in Kazakhstan (Karabalyk, Shortandy and Shagalaly) and one site in Russia (Omsk) in 2020-2022. The study aim was to identify markers demonstrating significant effects on agronomic traits. The average grain yield of individual trials varied from 118 to 569 g/m2. Grain yield was positively associated with the number of days to heading, plant height, number of grains per spike and 1000-kernel weight. Eight DNA markers demonstrated significant effects. The spring-type allele of the Vrn-A1 gene accelerated heading by two days (5.6%) and was present in 80% of the germplasm. The winter allele of the Vrn-A1 gene significantly increased grain yield by 2.7%. The late allele of the earliness marker per se, TaMOT1-D1, delayed development by 1.9% and increased yield by 4.5%. Translocation of 1B.1R was present in 21.8% of the material, which resulted in a 6.2% yield advantage compared to 1B.1B germplasm and a reduction in stem rust severity from 27.6 to 6.6%. The favorable allele of TaGS-D1 increased both kernel weight and yield by 2-3%. Four markers identified in ICARDA germplasm, ISBW2-GY (Kukri_c3243_1065, 3B), ISBW3-BM (TA004946-0577, 1B), ISBW10-SM2 (BS00076246_51, 5A), ISBW11-GY (wsnp_Ex_c12812_20324622, 4A), showed an improved yield in this study of 3-4%. The study recommends simultaneous validation and use of selected markers in KASIB's network.

Quality, Nutritional Properties, and Glycemic Index of Colored Whole Wheat Breads.

The main aim of this study was to investigate the quality and nutritional properties (dietary fiber, phenolic, antioxidant contents, and glycemic index) of breads made from whole wheat flours of colored wheats. White (cultivar Agronomicheskaya 5), red (Element 22), purple (EF 22 and Purple 8), and blue (Blue 10) colored wheats were used in the study. The whole wheat flours of Blue 10 and Purple 8 had higher farinograph stability, lower softening degree, and higher quality numbers indicating that they had better rheological properties. Bread produced from whole wheat flour of blue-colored grain had significantly higher loaf volume and better symmetry, crust color, crumb cell structure, and softness values among others (p < 0.05). The whole wheat bread produced using Element 22 had the highest crust and crumb L* color values, while Purple 8 and EF 22 had the lowest crust and crumb L* color values, suggesting that purple-colored grains have a tendency to make whole wheat bread with darker crust and crumb color. Bread produced from cultivar Blue 10 had the lowest firmness values while Element 22 had the highest firmness values. The highest total phenolic content and antioxidant capacity values were obtained from the whole wheat bread sample from purple-colored wheat (Purple 8). The whole wheat flour of Element 22 had the highest total dietary fiber content among all samples (p < 0.05). The differences between whole wheat bread samples in terms of total dietary fiber and glycemic index were not statistically significant. The results of the present study indicated that colored wheats can be used to produce whole wheat breads with higher nutritional properties and acceptable quality characteristics.

Phenolic Content and Antioxidant Capacity of Synthetic Hexaploid Wheats.

In this study, 21 synthetic hexaploid wheat samples were analyzed and compared for phenolic content (the Folin-Ciocalteu method), phenolic compositions, and antioxidant activity (DPPH, ABTS, and CUPRAC). The aim of the study was to determine the phenolic content and antioxidant activity of synthetic wheat lines developed from Ae. Tauschii, which has a wide genetic diversity, to be used in breeding programs for developing new varieties with better nutritional properties. Bound, free, and total phenolic contents (TPCs) of wheat samples were determined as 145.38-258.55 mg GAE/100 g wheat, 188.19-369.38 mg GAE/100 g wheat, and 333.58-576.93 mg GAE/100 g wheat, respectively. Phenolic compositions were detected by the HPLC system. Gallic acid was found in the highest concentrations in free fractions, whereas gallic, p-coumaric acid, and chlorogenic acid were generally found in the highest concentrations in bound fractions of the synthetic hexaploid wheat samples. The antioxidant activities (AA%) of the wheat samples were evaluated by the DPPH assay. AA% in the free extracts of the synthetic red wheat samples ranged from 33.0% to 40.5%, and AA% values in the bound extracts of the synthetic hexaploid wheat samples varied between 34.4% and 50.6%. ABTS and CUPRAC analyses were also used to measure antioxidant activities. The ABTS values of the free and bound extracts and total ABTS values of the synthetic wheat samples ranged from 27.31 to 123.18, 61.65 to 263.23, and 93.94 to 308.07 mg TE/100 g, respectively. The corresponding CUPRAC values of the synthetic wheats were between 25.78-160.94, 75.35-308.13, and 107.51-364.79 mg TE/100 g. This study revealed that synthetic hexaploid wheat samples are valuable resources for breeding programs for developing new wheat varieties with higher concentrations and better compositions of health-beneficial phytochemicals. The samples w1 (Ukr.-Od. 1530.94/Ae. squarrosa (629)), w18 (Ukr.-Od. 1530.94/Ae. squarrosa (1027)), and w20 (Ukr.-Od. 1530.94/Ae. squarrosa (392)) can be used as a genetic resource in breeding programs to enhance the nutritional quality of wheat.

Utilization of Intermediate Wheatgrass (Thinopyrum intermedium) as an Innovative Ingredient in Bread Making.

Intermediate wheatgrass (IWG; Thinopyrum intermedium), a nutritionally dense and sustainable crop, is a promising novel ingredient in bakery applications. The main aim of this study was to investigate the potential of IWG as a novel ingredient in breadmaking. The second aim was to investigate the characteristics of breads substituted with 15, 30, 45, and 60% IWG flour compared to control bread produced using wheat flour. The gluten content and quality, bread quality, bread staling, yellow pigment, and phenolic and antioxidant properties were determined. Enrichment with IWG flours significantly affected the gluten content and quality and bread characteristics. Increased levels of IWG flour substitution significantly decreased the Zeleny sedimentation and gluten index values and increased the dry and wet gluten contents. The bread yellow pigment content and crumb b* colour value increased with the increasing level of IWG supplementation. IWG addition also had a positive effect on the phenolic and antioxidant properties. Bread with 15% IWG substitution had the highest bread volume (485 mL) and lowest firmness values (654 g-force; g-f) compared to the other breads, including the control (i.e., wheat flour bread). The results indicated that IWG has great potential to be used in bread production as a novel, healthy, and sustainable ingredient.

Genetic Characterization of Spring Wheat Germplasm for Macro-, Microelements and Trace Metals.

Wheat as a staple food crop is the main source of micro- and macronutrients for most people of the world and is recognized as an attractive crop for biofortification. This study presents a comprehensive investigation of genomic regions governing grain micro- and macroelements concentrations in a panel of 135 diverse wheat accessions through a genome-wide association study. The genetic diversity panel was genotyped using the genotyping-by-sequencing (GBS) method and phenotyped in two environments during 2017−2018. Wide ranges of variation in nutrient element concentrations in grain were detected among the accessions. Based on 33,808 high-quality single nucleotide polymorphisms (SNPs), 2997 marker-element associations (MEAs) with −log10(p-value) > 3.5 were identified, representing all three subgenomes of wheat for 15-grain concentration elements. The highest numbers of MEAs were identified for Mg (499), followed by S (399), P (394), Ni (381), Cd (243), Ca (229), Mn (224), Zn (212), Sr (212), Cu (111), Rb (78), Fe (63), Mo (43), K (32) and Co (19). Further, MEAs associated with multiple elements and referred to as pleiotropic SNPs were identified for Mg, P, Cd, Mn, and Zn on chromosomes 1B, 2B, and 6B. Fifty MEAs were subjected to validation using KASIB multilocational trial at six sites in two years using 39 genotypes. Gene annotation of MEAs identified putative candidate genes that potentially encode different types of proteins related to disease, metal transportation, and metabolism. The MEAs identified in the present study could be potential targets for further validation and may be used in marker-assisted breeding to improve nutrient element concentrations in wheat grain.

Antioxidant Capacity and Profiles of Phenolic Acids in Various Genotypes of Purple Wheat.

The total phenolic content, phenolic compositions, and antioxidant capacity in the grain of 40 purple wheat genotypes were studied. In this study, purple wheats were investigated in terms of their composition of free and bound phenolic acids and 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity. The free phenolic content ranged from 164.25 to 271.05 mg GAE/100 g DW and the bound phenolic content was between 182.89-565.62 mg GAE/100 g wheat. The total phenolic content of purple wheat samples ranged from 352.65 to 771.83 mg GAE/100 g wheat. Gallic acid, protocatechuic acid, catechin, 4-hydroxybenzoic acid, syringic acid, ellagic acid, m-coumaric acid, o-coumaric acid, chrysin, caffeic acid, p-coumaric acid, ferulic acid, quercetin, kaempferol, rutin, sinapic acid, and chlorogenic acid were detected by HPLC system. Gallic acid, benzoic acid derivatives, and dominant phenolics, which are frequently found in cereals, were also dominant in purple wheat samples and were found in free fractions. The antioxidant capacity was assessed using the DPPH method. The antioxidant capacity (AA%) in the free phenolic extracts of the purple wheats was between 39.7% and 59.5%, and the AA% values of bound phenolic extract of the purple wheat varied between 42.6% and 62.7%. This study suggested that purple wheat samples have high phenolic compound content as antioxidant potential and therefore consumption of purple wheat-containing food products may provide health benefits.

Global genomic analyses of wheat powdery mildew reveal association of pathogen spread with historical human migration and trade.

The fungus Blumeria graminis f. sp. tritici causes wheat powdery mildew disease. Here, we study its spread and evolution by analyzing a global sample of 172 mildew genomes. Our analyses show that B.g. tritici emerged in the Fertile Crescent during wheat domestication. After it spread throughout Eurasia, colonization brought it to America, where it hybridized with unknown grass mildew species. Recent trade brought USA strains to Japan, and European strains to China. In both places, they hybridized with local ancestral strains. Thus, although mildew spreads by wind regionally, our results indicate that humans drove its global spread throughout history and that mildew rapidly evolved through hybridization.

Wheat Stem Rust Back in Europe: Diversity, Prevalence and Impact on Host Resistance.

The objective of this study was to investigate the re-emergence of a previously important crop pathogen in Europe, Puccinia graminis f.sp. tritici, causing wheat stem rust. The pathogen has been insignificant in Europe for more than 60 years, but since 2016 it has caused epidemics on both durum wheat and bread wheat in local areas in southern Europe, and additional outbreaks in Central- and West Europe. The prevalence of three distinct genotypes/races in many areas, Clade III-B (TTRTF), Clade IV-B (TKTTF) and Clade IV-F (TKKTF), suggested clonal reproduction and evolution by mutation within these. None of these genetic groups and races, which likely originated from exotic incursions, were detected in Europe prior to 2016. A fourth genetic group, Clade VIII, detected in Germany (2013), was observed in several years in Central- and East Europe. Tests of representative European wheat varieties with prevalent races revealed high level of susceptibility. In contrast, high diversity with respect to virulence and Simple Sequence Repeat (SSR) markers were detected in local populations on cereals and grasses in proximity to Berberis species in Spain and Sweden, indicating that the alternate host may return as functional component of the epidemiology of wheat stem rust in Europe. A geographically distant population from Omsk and Novosibirsk in western Siberia (Russia) also revealed high genetic diversity, but clearly different from current European populations. The presence of Sr31-virulence in multiple and highly diverse races in local populations in Spain and Siberia stress that virulence may emerge independently when large geographical areas and time spans are considered and that Sr31-virulence is not unique to Ug99. All isolates of the Spanish populations, collected from wheat, rye and grass species, were succesfully recovered on wheat, which underline the plasticity of host barriers within P. graminis. The study demonstrated successful alignment of two genotyping approaches and race phenotyping methodologies employed by different laboratories, which also allowed us to line up with previous European and international studies of wheat stem rust. Our results suggest new initiatives within disease surveillance, epidemiological research and resistance breeding to meet current and future challenges by wheat stem rust in Europe and beyond.

Variation of Macro- and Microelements, and Trace Metals in Spring Wheat Genetic Resources in Siberia.

Western Siberia is one of the major spring wheat regions of Russia, cultivating over 7 Mha. The objective of the study was to evaluate the variation of macro- and microelements, and of trace metals in four distinct groups of genetic resources: primary synthetics from CIMMYT (37 entries), primary synthetics from Japan (8), US hard red spring wheat cultivars (14), and material from the Kazakhstan-Siberian Network on Spring Wheat Improvement (KASIB) (74). The experiment was conducted at Omsk State Agrarian University, using a random complete block design with four replicates in 2017 and 2018. Concentrations of 15 elements were included in the analysis: macroelements, Ca, K, Mg, P, and S; microelements, Fe, Cu, Mn, and Zn; toxic trace elements, Cd, Co, Ni; and trace elements, Mo, Rb, and Sr. Protein content was found to be positively correlated with the concentrations of 11 of the elements in one or both years. Multiple regression was used to adjust the concentration of each element, based on significant correlations with agronomic traits and macroelements. All 15 elements were evaluated for their suitability for genetic enhancement, considering phenotypic variation, their share of the genetic component in this variation, as well as the dependence of the element concentration on other traits. Three trace elements (Sr, Mo, and Co) were identified as traits that were relatively easy to enhance through breeding. These were followed by Ca, Cd, Rb, and K. The important biofortification elements Mn and Zn were among the traits that were difficult to enhance genetically. The CIMMYT and Japanese synthetics had significantly higher concentrations of K and Sr, compared to the local check. The Japanese synthetics also had the highest concentrations of Ca, S, Cd, and Mo. The US cultivars had concentrations of Ca as high as the Japanese synthetics, and the highest concentrations of Mg and Fe. KASIB's germplasm had near-average values for most elements. Superior germplasm, with high macro- and microelement concentrations and low trace-element concentrations, was found in all groups of material included.

Marker-Trait Associations for Enhancing Agronomic Performance, Disease Resistance, and Grain Quality in Synthetic and Bread Wheat Accessions in Western Siberia.

Exploiting genetically diverse lines to identify genes for improving crop performance is needed to ensure global food security. A genome-wide association study (GWAS) was conducted using 46,268 SNP markers on a diverse panel of 143 hexaploid bread and synthetic wheat to identify potential genes/genomic regions controlling agronomic performance (yield and 26 yield-related traits), disease resistance, and grain quality traits. From phenotypic evaluation, we found large genetic variation among the 35 traits and recommended five lines having a high yield, better quality, and multiple disease resistance for direct use in a breeding program. From a GWAS, we identified a total of 243 significant marker-trait associations (MTAs) for 35 traits that explained up to 25% of the phenotypic variance. Of these, 120 MTAs have not been reported in the literature and are potentially novel MTAs. In silico gene annotation analysis identified 116 MTAs within genes and of which, 21 MTAs were annotated as a missense variant. Furthermore, we were able to identify 23 co-located multi-trait MTAs that were also phenotypically correlated to each other, showing the possibility of simultaneous improvement of these traits. Additionally, most of the co-located MTAs were within genes. We have provided genomic fingerprinting for significant markers with favorable and unfavorable alleles in the diverse set of lines for developing elite breeding lines from useful trait-integration. The results from this study provided a further understanding of genetically complex traits and would facilitate the use of diverse wheat accessions for improving multiple traits in an elite wheat breeding program.

Genetic diversity and population structure analysis of synthetic and bread wheat accessions in Western Siberia.

Recurrent selection and intercrossing between best of the best parents in each generation of breeding cycle resulted in a narrower genetic diversity in elite wheat (Triticum aestivum L.) germplasm. Therefore, we investigated diverse source of 143 synthetic and bread wheat accessions for identifying potentially rich genetic resources for improving the genetic diversity in wheat. This study identified 47,526 genotyping-by-sequencing-derived SNP markers that were nearly evenly distributed across three genomes of wheat. The population structure analysis identified three distinct clusters (Japan synthetics, CIMMYT synthetics, and bread wheat) of wheat genotypes on the basis of type and geographical origin of wheat accessions. Population differentiation using analysis of molecular variance indicated 21% of the total genetic variance among subgroups and the remainder within subgroups. This study also identified that the Japan synthetic group was the most divergent group compared with other subgroups. The genetic diversity comparisons between synthetic and bread wheat lines showed that the gene diversity of synthetic wheat was 33% higher than bread wheat accessions, indicating the potential use of these lines for broadening the genetic diversity of modern wheat cultivars. The results from this study will be helpful in further understanding genomic features of wheat and facilitate their use in wheat breeding programs.

Primary hexaploid synthetics: Novel sources of wheat disease resistance.

Climate change is leading to increased occurrence of and yield losses to wheat diseases. Managing these diseases by introducing new, effective and diverse resistance genes into cultivars represents an important component of sustainable wheat production. In 2016 and 2017 a set of primary hexaploid synthetic wheat was studied under high disease pressure: powdery mildew, leaf and stem rust in Omsk; Septoria tritici and S. nodorum in Moscow. A total of 28 synthetics (19 CIMMYT synthetics and 9 Japanese synthetics) were selected as having combined resistance to at least two diseases in both years of testing. Two synthetics (entries 13 and 18) originating from crosses between winter durum wheat Ukrainka odesskaya-1530.94 and various Aegilopes taushii accessions, and four synthetics (entries 20, 21, 23 and 24) from cross between Canadian durum wheat Langdon and Ae. taushii were resistant to all four pathogens. Pathological and molecular markers evaluation of resistance suggests presence of new genes and diverse types of resistance. The novel genetic sources of disease resistance identified in this study can be successfully utilized in wheat breeding.

Effect of climate change on spring wheat yields in North America and Eurasia in 1981-2015 and implications for breeding.

Wheat yield dynamic in Canada, USA, Russia and Kazakhstan from 1981 till 2015 was related to air temperature and precipitation during wheat season to evaluate the effects of climate change. The study used yield data from the provinces, states and regions and average yield from 19 spring wheat breeding/research sites. Both at production and research sites grain yield in Eurasia was two times lower compared to North America. The yearly variations in grain yield in North America and Eurasia did not correlate suggesting that higher yield in one region was normally associated with lower yield in another region. Minimum and maximum air temperature during the wheat growing season (April-August) had tendency to increase. While precipitation in April-August increased in North American sites from 289 mm in 1981-1990 to 338 mm in 2006-2015 it remained constant and low at Eurasian sites (230 and 238 mm, respectively). High temperature in June and July negatively affected grain yield in most of the sites at both continents. Climatic changes resulted in substantial changes in the dates of planting and harvesting normally leading to extension of growing season. Longer planting-harvesting period was positively associated with the grain yield for most of the locations. The climatic changes since 1981 and spring wheat responses suggest several implications for breeding. Gradual warming extends the wheat growing season and new varieties need to match this to utilize their potential. Higher rainfall during the wheat season, especially in North America, will require varieties with higher yield potential responding to moisture availability. June is a critical month for spring wheat in both regions due to the significant negative correlation of grain yield with maximum temperature and positive correlation with precipitation. Breeding for adaptation to higher temperatures during this period is an important strategy to increase yield.

The \textit{BRAF} V600E mutation in single- institution study of Russian melanoma patients.

BACKGROUND: The activating mutation BRAF V600E is considered to be a diagnostic cutaneous melanoma (CM) marker important for prognosis and targeted therapy. OBJECTIVE: The aim of this study was to determine the frequency of the V600E mutation in CM patients in Russia and to estimate the influence of the BRAF gene mutation status on prognosis and clinical outcome. METHODS: To ensure mutation detection in FFPE tissue, interlaboratory validation was performed using three different methods: allele-specific hybridisation on a biochip, allele-specific real-time PCR and, in some cases, direct sequencing. RESULTS: Mutation V600E was detected in 49% of patients. The age of disease manifestation was significantly lower in mutated (MT) BRAF patients, and the median age difference between the wild-type (WT) and MT BRAF groups (P= 0.002) was 10 years. A tumour thickness more than 1 mm was also more frequently observed in the MT BRAF group (P= 0.059). Patients from the MT BRAF group were more likely to have ulceration compared to the WT group (P= 0.088). No statistically significant differences were found between the relapse-free, progression-free or overall survival of CM patients in the MT BRAF and WT BRAF groups. CONCLUSIONS: The data obtained show that the V600E BRAF mutation occurred in about half of melanoma patients; it was associated with earlier manifestation of melanoma and likely with more aggressive clinical features.

Sensitive allele-specific real-time PCR test for mutations in BRAF codon V600 in skin melanoma.

Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.

Valproate and bone loss: iTRAQ proteomics show that valproate reduces collagens and osteonectin in SMA cells.

Valproate is commonly used as an anticonvulsant and mood stabilizer, but its long-term side-effects can include bone loss. As a histone deacetylase (HDAC) inhibitor, valproate has also been considered for treatment of spinal muscular atrophy (SMA). Using iTRAQ labeling technology, followed by two-dimensional liquid chromatography and mass spectrometry analysis, a quantitative comparison of the proteome of an SMA cell line, with and without valproate treatment, was performed. The most striking change was a reduction in collagens I and VI, while over 1000 other proteins remained unchanged. The collagen I alpha-chain precursor was also reduced by more than 50% suggesting that valproate affects collagen I synthesis. The collagen-binding glycoprotein, osteonectin (SPARC, BM-40) was one of the few other proteins that were significantly reduced by valproate treatment. Collagen I is the main protein component of bone matrix and osteonectin has a major role in bone development, so the results suggest a possible molecular mechanism for bone loss following long-term exposure to valproate. SMA patients may already suffer bone weakness as a result of SMN1 gene deletion, so further bone loss would be undesirable.

hAda3 degradation by papillomavirus type 16 E6 correlates with abrogation of the p14ARF-p53 pathway and efficient immortalization of human mammary epithelial cells.

Two activities of human papillomavirus type 16 E6 (HPV16 E6) are proposed to contribute to the efficient immortalization of human epithelial cells: the degradation of p53 protein and the induction of telomerase. However, the requirement for p53 inactivation has been debated. Another E6 target is the hAda3 protein, a p53 coactivator and a component of histone acetyltransferase complexes. We have previously described the role of hAda3 and p53 acetylation in p14ARF-induced human mammary epithelial cell (MEC) senescence (P. Sekaric, V. A. Shamanin, J. Luo, and E. J. Androphy, Oncogene 26:6261-6268, 2007). In this study, we analyzed a set of HPV16 E6 mutants for the ability to induce hAda3 degradation. E6 mutants that degrade hAda3 but not p53 could abrogate p14ARF-induced growth arrest despite the presence of normal levels of p53 and efficiently immortalized MECs. However, two E6 mutants that previously were reported to immortalize MECs with low efficiency were found to be defective for both p53 and hAda3 degradation. We found that these immortal MECs select for reduced p53 protein levels through a proteasome-dependent mechanism. The findings strongly imply that the inactivation of the p14ARF-p53 pathway, either by the E6-mediated degradation of p53 or hAda3 or by cellular adaptation, is required for MEC immortalization.

Immortalization of human mammary epithelial cells is associated with inactivation of the p14ARF-p53 pathway.

Inactivation of the ARF-p53 tumor suppressor pathway leads to immortalization of murine fibroblasts. The role of this pathway in immortalization of human epithelial cells is not clear. We analyzed the functionality of the p14(ARF)-p53 pathway in human mammary epithelial cells (MEC) immortalized by human papillomavirus 16 (HPV16) E6, the p53 degradation-defective E6 mutant Y54D, or hTERT. E6-MEC or E6Y54D-MEC maintains high-level expression of p14(ARF). Late-passage hTERT-immortalized MEC express p53 but down-regulate p14(ARF). Enforced expression of p14(ARF) induces p53-dependent senescence in hTERT-MEC, while both E6-MEC and E6Y54D-MEC are resistant. We show that E6Y54D inhibits p14(ARF)-induced activation of p53 without inactivation of the p53-dependent DNA damage response. Hence, p53 degradation and inhibition of p14(ARF) signaling to p53 are independent functions of HPV16 E6. Our observations imply that long-term proliferation of MEC requires inactivation of the p14(ARF)-p53 pathway.