A Systematic Review of the 2016 National Academy of Engineering Exemplary Ethics Programs: Revisions to a Coding Framework.

Engineering ethics is a required aspect of accredited ABET programs, but there is widespread variation in how ethics is taught, to what ends, and how those ends are assessed. This variation makes it challenging to identify practices for teaching ethics to engineers aligned with extant practices in the field. In this study, we revise a recent coding framework by reviewing exemplary engineering ethics programs recognized by the National Academy of Engineering in 2016, or what we refer to as "exemplars." We pursue two primary objectives: (1) To apply and revise a prior coding framework to codify ethics learning objectives, instructional strategies, and assessment strategies in engineering education; and (2) To use the revised coding framework to identify trends in learning objectives, instructional strategies, and assessment strategies of NAE exemplars. We employ systemic review procedures to update the coding framework using 24 of 25 exemplars as a data source. The updated framework includes four primary categories associated with learning objectives, instructional strategies, assessment data collection strategies, and assessment design characteristics. Results indicate that ethical sensitivity or awareness was present in every exemplar as a learning objective, often alongside ethical reasoning-based learning objectives and the formation of professional skills. Exemplars employed numerous instructional strategies in tandem, as we coded eight out of 18 instructional strategies among at least half of the exemplars. Assignments/homework and summative reflections were the most oft-used sources of assessment data. Due to our challenges in coding assessment approaches, we offer practical suggestions for assessing engineering ethics instruction which are based on many of our coding discussions. We hope that this coding framework, the results classifying exemplary features of the NAE programs, and our practical suggestions can guide future instructors as they design, classify, assess, and report their approaches to engineering ethics education.

Single-cell analysis of habituation in Stentor coeruleus.

Although learning is often viewed as a unique feature of organisms with complex nervous systems, single-celled organisms also demonstrate basic forms of learning. The giant ciliate Stentor coeruleus responds to mechanical stimuli by contracting into a compact shape, presumably as a defense mechanism. When a Stentor cell is repeatedly stimulated at a constant level of force, it will learn to ignore that stimulus but will still respond to stronger stimuli. Prior studies of habituation in Stentor reported a graded response, suggesting that cells transition through a continuous range of response probabilities. By analyzing single cells using an automated apparatus to deliver calibrated stimuli, we find that habituation occurs via a single step-like switch in contraction probability within each cell, with the graded response in a population arising from the random distribution of switching times in individual cells. This step-like response allows Stentor behavior to be represented by a simple two-state model whose parameters can be estimated from experimental measurements. We find that transition rates depend on stimulus force and also on the time between stimuli. The ability to measure the behavior of the same cell to the same stimulus allowed us to quantify the functional heterogeneity among single cells. Together, our results suggest that the behavior of Stentor is governed by a two-state stochastic machine whose transition rates are sensitive to the time series properties of the input stimuli.

Modular, cascade-like transcriptional program of regeneration in Stentor.

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis in a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half regenerates an intact cell. We used RNA sequencing (RNAseq) to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA -binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes. By comparing transcriptional profiles of different regeneration events, we identified distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression after blocking translation, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the RNA-binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi-mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells.

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell.1-4 Patterning within cells can extend down to the level of individual proteins and mRNA.5,6 But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation7-11 has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast12-15 found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning-the giant ciliate Stentor coeruleus.16,17 Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning.1,18 A Stentor cell is roughly 2 mm long, allowing a "proteomic dissection" in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell's anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

Rsf-1, a chromatin remodeling protein, induces DNA damage and promotes genomic instability.

Rsf-1 (HBXAP) has been reported as an amplified gene in human cancer, including the highly aggressive ovarian serous carcinoma. Rsf-1 protein interacts with SNF2H to form an ISWI chromatin remodeling complex, RSF. In this study, we investigated the functional role of Rsf-1 by observing phenotypes after expressing it in nontransformed cells. Acute expression of Rsf-1 resulted in DNA damage as evidenced by DNA strand breaks, nuclear γH2AX foci, and activation of the ATM-CHK2-p53-p21 pathway, leading to growth arrest and apoptosis. Deletion mutation and gene knockdown assays revealed that formation of a functional RSF complex with SNF2H was required for Rsf-1 to trigger DNA damage response (DDR). Gene knock-out of TP53 alleles, TP53 mutation, or treatment with an ATM inhibitor abolished up-regulation of p53 and p21 and prevented Rsf-1-induced growth arrest. Chronic induction of Rsf-1 expression resulted in chromosomal aberration and clonal selection for cells with c-myc amplification and CDKN2A/B deletion. Co-culture assays indicated Rsf-1-induced DDR as a selecting barrier that favored outgrowth of cell clones with a TP53 mutation. The above findings suggest that increased Rsf-1 expression and thus excessive RSF activity, which occurs in tumors harboring Rsf-1 amplification, can induce chromosomal instability likely through DDR.

Analysis of Motility Patterns of Stentor During and After Oral Apparatus Regeneration Using Cell Tracking.

Stentor coeruleus is a well-known model organism for the study of unicellular regeneration. Transcriptomic analysis of individual cells revealed hundreds of genes-many not associated with the oral apparatus (OA)-that are differentially regulated in phases throughout the regeneration process. It was hypothesized that this systemic reorganization and mobilization of cellular resources towards growth of a new OA will lead to observable changes in movement and behavior corresponding in time to the phases of differential gene expression. However, the morphological complexity of S. coeruleus necessitated the development of an assay to capture the statistics and timescale. A custom script was used to track cells in short videos, and statistics were compiled over a large population (N ~100). Upon loss of the OA, S. coeruleus initially loses the ability for directed motion; then starting at ~4 h, it exhibits a significant drop in speed until ~8 h. This assay provides a useful tool for the screening of motility phenotypes and can be adapted for the investigation of other organisms.

Aurora kinase inhibitors delay regeneration in Stentor coeruleus at an intermediate step.

The giant unicellular ciliate Stentor coeruleus can be cut into pieces and each piece will regenerate into a healthy, full-sized individual. The molecular mechanism for how Stentor regenerates is a complete mystery, however, the process of regeneration shows striking similarities to the process of cell division. On a morphological level, the process of creating a second mouth in division or a new oral apparatus in regeneration have the same steps and occur in the same order. On the transcriptional level, genes encoding elements of the cell division and cell cycle regulatory machinery, including Aurora kinases, are differentially expressed during regeneration. This suggests that there may be some common regulatory mechanisms involved in both regeneration and cell division. If the cell cycle machinery really does play a role in regeneration, then inhibition of proteins that regulate the timing of cell division may also affect the timing of regeneration in Stentor. Here we show that two well-characterized Aurora kinase A+B inhibitors that affect the timing of regeneration. ZM447439 slows down regeneration by at least one hour. PF03814735 completely suppresses regeneration until the drug is removed. Here we provide the first direct experimental evidence that Stentor may harness the cell division machinery to regulate the sequential process of regeneration.

Cancer cells and normal cells differ in their requirements for Thoc1.

The evolutionarily conserved TREX (Transcription/Export) complex physically couples transcription, messenger ribonucleoprotein particle biogenesis, RNA processing, and RNA export for a subset of genes. HPR1 encodes an essential component of the S. cerevisiae TREX complex. HPR1 loss compromises transcriptional elongation, nuclear RNA export, and genome stability. Yet, HPR1 is not required for yeast viability. Thoc1 is the recently discovered human functional orthologue of HPR1. Thoc1 is expressed at higher levels in breast cancer than in normal epithelia, and expression levels correlate with tumor size and metastatic potential. Depletion of Thoc1 protein (pThoc1) in human cancer cell lines compromises cell proliferation. It is currently unclear whether Thoc1 is essential for all mammalian cells or whether cancer cells may differ from normal cells in their dependence on Thoc1. To address this issue, we have compared the requirements for Thoc1 in the proliferation and survival of isogenic normal and oncogene-transformed cells. Neoplastic cells rapidly lose viability via apoptotic cell death on depletion of pThoc1. Induction of apoptotic cell death is coincident with increased DNA damage as indicated by the appearance of phosphorylated histone H2AX. In contrast, the viability of normal cells is largely unaffected by pThoc1 loss. Normal cells lacking Thoc1 cannot be transformed by forced expression of E1A and Ha-ras, suggesting that Thoc1 may be important for neoplastic transformation. In sum, our data are consistent with the hypothesis that cancer cells require higher levels of pThoc1 for survival than normal cells. If true, pThoc1 may provide a novel molecular target for cancer therapy.

Knowledge and awareness of cervical cancer in Southwestern Ethiopia is lacking: A descriptive analysis.

PURPOSE: Cervical cancer remains the second most common cancer and cancer-related death among women in Ethiopia. This is the first study, to our knowledge, describing the demographic, and clinicopathologic characteristics of cervical cancer cases in a mainly rural, Southwestern Ethiopian population with a low literacy rate to provide data on the cervical cancer burden and help guide future prevention and intervention efforts. METHODS: A descriptive analysis of 154 cervical cancer cases at the Jimma University Teaching Hospital in Southwestern Ethiopia from January 2008 -December 2010 was performed. Demographic and clinical characteristics were obtained from patient questionnaires and cervical punch biopsies were histologically examined. RESULTS: Of the 154 participants with a histopathologic diagnosis of cervical cancer, 95.36% had not heard of cervical cancer and 89.6% were locally advanced at the time of diagnosis. Moreover, 86.4% of participants were illiterate, and 62% lived in a rural area. CONCLUSION: A majority of the 154 women with cervical cancer studied at the Jimma University Teaching Hospital in Southwestern Ethiopia were illiterate, had not heard of cervical cancer and had advanced disease at the time of diagnosis. Given the low rates of literacy and knowledge regarding cervical cancer in this population which has been shown to correlate with a decreased odds of undergoing screening, future interventions to address the cervical cancer burden here must include an effective educational component.

Methods for the Study of Regeneration in Stentor.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Oncogenic ras and p53 cooperate to induce cellular senescence.

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.

Molecular signature of oncogenic ras-induced senescence.

Senescence irreversibly arrests the proliferation of cells that have sustained significant cellular stress. Replicative senescence, due to the shortening and dysfunction of telomeres, appears to provide a barrier to the immortalization of cells and development of cancer. In normal human fibroblasts, senescence induced by oncogenic H-ras displays a nearly identical cellular phenotype to that of replicative senescence, suggesting the activation of a common senescence mechanism. In this study, we investigated the gene expression profile of oncogenic H-ras-induced senescent human diploid fibroblasts. We found altered gene expression of various cell cycle regulators in both oncogenic H-ras-induced senescent cells and replicative senescent cells. Similar to replicative senescent cells, H-ras-induced senescent cells exhibited specific downregulation of genes involved in G2/M checkpoint control and contained tetraploid cells that were arrested in a G1 state. This observation suggests that the inactivation of G2/M checkpoints may be involved in senescence and may play a role in the generation of senescent G1 tetraploid cells. Lastly, we have identified two genes, topoisomerase IIalpha and HDAC9, whose expression was specifically altered under several conditions associated with senescence, suggesting that these two molecules may be novel biomarkers for senescent human fibroblasts.

Retroviral expression of human cystatin genes in HeLa cells.

Retroviral gene transfer is a highly efficient and effective method of stably introducing genetic material into the genome of specific cell types. The process involves the transfection of retroviral expression vectors into a packaging cell line, the isolation of viral particles, and the infection of target cell lines. Compared to traditional gene transfer methods such as liposome-mediated transfection, retroviral gene transfer allows for stable gene expression in cell populations without the need for lengthy selection and cloning procedures. This is particularly helpful when studying gene products that have negative effect on cell growth and viability. Here, we describe the retroviral transfer of cystatin cDNAs using HEK293-derived Phoenix packaging cells and human HeLa cervical carcinoma cells as target cells.

Thermal transport characteristics of human skin measured in vivo using ultrathin conformal arrays of thermal sensors and actuators.

Measurements of the thermal transport properties of the skin can reveal changes in physical and chemical states of relevance to dermatological health, skin structure and activity, thermoregulation and other aspects of human physiology. Existing methods for in vivo evaluations demand complex systems for laser heating and infrared thermography, or they require rigid, invasive probes; neither can apply to arbitrary regions of the body, offers modes for rapid spatial mapping, or enables continuous monitoring outside of laboratory settings. Here we describe human clinical studies using mechanically soft arrays of thermal actuators and sensors that laminate onto the skin to provide rapid, quantitative in vivo determination of both the thermal conductivity and thermal diffusivity, in a completely non-invasive manner. Comprehensive analysis of measurements on six different body locations of each of twenty-five human subjects reveal systematic variations and directional anisotropies in the characteristics, with correlations to the thicknesses of the epidermis (EP) and stratum corneum (SC) determined by optical coherence tomography, and to the water content assessed by electrical impedance based measurements. Multivariate statistical analysis establishes four distinct locations across the body that exhibit different physical properties: heel, cheek, palm, and wrist/volar forearm/dorsal forearm. The data also demonstrate that thermal transport correlates negatively with SC and EP thickness and positively with water content, with a strength of correlation that varies from region to region, e.g., stronger in the palmar than in the follicular regions.

Mating of the stichotrichous ciliate Oxytricha trifallax induces production of a class of 27 nt small RNAs derived from the parental macronucleus.

Ciliated protozoans possess two types of nuclei; a transcriptionally silent micronucleus, which serves as the germ line nucleus, and a transcriptionally active macronucleus, which serves as the somatic nucleus. The macronucleus is derived from a new diploid micronucleus after mating, with epigenetic information contributed by the parental macronucleus serving to guide the formation of the new macronucleus. In the stichotrichous ciliate Oxytricha trifallax, the macronuclear DNA is highly processed to yield gene-sized nanochromosomes with telomeres at each end. Here we report that soon after mating of Oxytricha trifallax, abundant 27 nt small RNAs are produced that are not present prior to mating. We performed next generation sequencing of Oxytricha small RNAs from vegetative and mating cells. Using sequence comparisons between macronuclear and micronuclear versions of genes, we found that the 27 nt RNA class derives from the parental macronucleus, not the developing macronucleus. These small RNAs are produced equally from both strands of macronuclear nanochromosomes, but in a highly non-uniform distribution along the length of the nanochromosome, and with a particular depletion in the 30 nt telomere-proximal positions. This production of small RNAs from the parental macronucleus during macronuclear development stands in contrast to the mechanism of epigenetic control in the distantly related ciliate Tetrahymena. In that species, 28-29 nt scanRNAs are produced from the micronucleus and these micronuclear-derived RNAs serve as epigenetic controllers of macronuclear development. Unlike the Tetrahymena scanRNAs, the Oxytricha macronuclear-derived 27 mers are not modified by 2'O-methylation at their 3' ends. We propose models for the role of these "27macRNAs" in macronuclear development.

Novel expression of CST1 as candidate senescence marker.

Senescent cells exhibit altered expression of numerous genes. Identifying the significance of the changes in gene expression may help advance our understanding of the senescence biology. Here, we report on the consistent and strong upregulation of CST1 expression during cellular senescence, independent of the initial trigger. CST1 expression at both the messenger RNA and protein levels was barely detected in control cells, which included early passage proliferating, quiescent, or immortal human fibroblasts and various human tumor cell lines. Immunoblotting and immunofluorescence cytochemical studies further suggest that CST1 accumulates intracellularly, within vesicular structures. We discuss these results in light of the known function of CST1 as a potent inhibitor of lysosomal cysteine proteases.

Restoration of immune response gene induction in trophoblast tumor cells associated with cellular senescence.

Trophoblast cells and many cancer cells that harbor foreign antigens may evade immunity by epigenetic silencing of key immune response genes, including MHC class I and II and CD40. Chromatin active agents, such as histone deacetylase inhibitors (HDACi), induce immune response gene expression but often the expression levels are low and the cells lack a robust antigen presentation response. We show here that pre-treatment of trophoblast cells and certain cancer cells with agents that activate stress pathways (Ras oncogene, PMA or H2O2) and induce senescence can substantially enhance the induction of immune response genes (MHC class II, CD40, MICA, MICB) by HDACi and restore a vigorous IFN-gamma response in trophoblast cells and tumor cells. These results could potentially impact the development of novel anti-cancer therapeutic strategies.

Dissecting the senescence-like program in tumor cells activated by Ras signaling.

Activated Ras signaling can induce a permanent growth arrest in osteosarcoma cells. Here, we report that a senescence-like growth inhibition is also achieved in human carcinoma cells upon the transduction of H-Ras(V12). Ras-induced tumor senescence can be recapitulated by the transduction of activated, but not wild-type, MEK. The ability for H-Ras(V12) to suppress tumor cell growth is drastically compromised in cells that harbor endogenous activating ras mutations. Notably, growth inhibition of tumor cells containing ras mutations can be achieved through the introduction of activated MEK. Tumor senescence induced by Ras signaling can occur in the absence of p16 or Rb and is not interrupted by the inactivation of Rb, p107, or p130 via short hairpin RNA or the transduction with HPV16 E7. In contrast, inactivation of p21 via short hairpin RNA disrupts Ras-induced tumor senescence. In summary, this study uncovers a senescence-like program activated by Ras signaling to inhibit cancer cell growth. This program appears to be intact in cancer cells that do not harbor ras mutations. Moreover, cancer cells that carry ras mutations remain susceptible to tumor senescence induced by activated MEK. These novel findings can potentially lead to the development of innovative cancer intervention.

Defined genetic events associated with the spontaneous in vitro transformation of ElA/Ras-expressing human IMR90 fibroblasts.

In contrast to rodent cells, normal human fibroblasts are generally resistant to neoplastic transformation in vitro. Here, we report the derivation and characterization of a spontaneously transformed cell line from normal human IMR90 fibroblasts transduced with E1A and Ras oncogenes. Unlike the parental, non-tumorigenic E1A/Ras-expressing IMR90 cells, these spontaneously transformed cells displayed aberrant growth potential in vitro and were capable of tumorigenesis in vivo. In contrast to the parental E1A/Ras-expressing cells, both the spontaneously transformed cells and cells derived from resultant tumors displayed specific t(7q;8q) and t(5q;17) structural chromosomal changes. Chromosome 8q contains c-Myc, which is capable of activating the telomerase catalytic subunit hTERT. Notably, upregulation of c-Myc, hTERT and telomerase activity were detected only in the tumorigenic cells. Transduction of Myc siRNA into the tumorigenic cells led to a concomitant downregulation of hTERT. Furthermore, transduction of Myc or hTERT into the non-tumorigenic E1A/Ras-expressing IMR90 cells was able to confer tumorigenesis on these cells. These studies suggest that the t(7;8) translocation may result in Myc overexpression and its subsequent activation of hTERT, which may contribute to the tumorigenicity of the IMR90 cells. Furthermore, this report describes additional successful neoplastic transformation of human IMR90 fibroblasts by defined genetic elements. The spontaneously transformed cells we have derived provide a valuable model system for the study of neoplastic transformation.

Expression of p16INK4A variants in senescent human fibroblasts independent of protein phosphorylation.

Upregulation of the p16 tumor suppressor is a hallmark of senescence in human fibroblasts. In this study, we investigated potential protein modification of p16 in senescent human fibroblasts using 2D SDS-PAGE analysis. Three distinct p16 variants with isoelectric points of 5.2, 5.4, and 5.6, were consistently detected in normal human IMR90 fibroblasts that had undergone senescence due to forced expression of oncogenic H-ras or culture passage. Moreover, in contrast to short-term serum starvation, which induces quiescence, IMR90 fibroblasts cultured in low serum for a prolonged period exhibited senescent phenotypes and expression of the three p16 variants. All three p16 variants are unlikely phosphoproteins since they failed to react with antibodies against phospho-serine, and were resistant to the treatment with phosphatases. Functionally, co-immunoprecipitation assays using antibodies against cdk4 and/or cdk6 revealed that only the two most acidic p16 variants associated with cdk4/6. Moreover, senescence induced by the forced expression of p16 in early passage IMR90 fibroblasts or osteosarcoma U2OS cells was accompanied by expression of the two most acidic p16 variants, which also associated with cdk4/6. In summary, we report that prolonged serum starvation-induced senescence may provide an additional model for studying biochemical changes in senescence, including p16 regulation. Furthermore, induction of endogenous p16 in senescent human fibroblasts correlates with the expression of three distinct p16 variants independent of protein phosphorylation. Lastly, expression of the two cdk-bound variants is sufficient to induce senescence in human cells.