Epistemic Peerhood as a Model To Improve Gender-Affirming Care in Medical Education.

Issue: Inadequate training around gender-affirming care is a critical gap in health care and medical education that causes disparities and leads to injury for transgender, nonbinary, and other gender-diverse patients. In contrast to this widespread provider knowledge gap, gender-diverse patients bring critical knowledge from their own experiences to health care. Embracing varied epistemologies, or sources of knowledge, within medical education has the potential to enhance gender-affirming care by intentionally placing value on the lived experiences and emphasizing the credibility of gender-diverse patients. Evidence: In this article, the authors endorse a model of epistemic peerhood in which the embodied knowledge of gender-diverse patients and the authoritative knowledge of providers are each valued for their contribution to care. The authors reflect on experiences developing gender-affirming healthcare curricula and how medical education has not yet adequately addressed gender-diverse care without embracing community knowledge. Implications: The authors identify three vital areas to integrate epistemic peerhood in medical training to address gaps in gender-affirming care: (1) collaborative student training methods that reflect embodied knowledge in the absence of, or in addition to, clinical expertise on gender-affirming care; (2) sustainable partnerships between academic programs and gender-diverse communities that foster continuous engagement from collaborators with lived experience; and (3) broad community input about best practices for representing gender diversity in patient simulation. Embracing epistemic peerhood in each of these areas would result in broader gender-diverse community representation and leadership in medical education, which would ultimately strengthen gender-affirming healthcare training.

Perspectives of Transgender and Genderqueer Standardized Patients.

Phenomenon: Disparities in health and healthcare for gender minorities (GMs) such as transgender people are significant, and medical educators have a responsibility to ensure trainees master the clinical skills required to provide them with quality care. We implemented a standardized patient (SP) scenario designed to measure students' ability to provide gender-affirming care and sought to understand the experiences and perceptions of the GMs who served as SPs in this case. Our key research question was: how do GM SPs describe the experience of serving as an SP on a gender-affirming care clinical case? Approach: Semi-structured focus groups were conducted with GM SPs (n = 10) to understand their experiences and gauge their perceptions of portraying a patient seeking gender-affirming care. The patient they portrayed matched their own gender identity. Focus groups were transcribed verbatim and analyzed using inductive thematic analysis. Findings: We developed three primary themes in our analysis: personal connection, gap identification, and insight into medical education. The SPs reported a personal connection to this case, enabling them to give nuanced feedback, confront bias they encountered, and foster connection to their broader community. They were able to identify specific gaps related to communication skills, assumptions, and knowledge about gender identity and gender-affirming care. They gained valuable insight into medical education such as the complexity of learning clinical skills and roadblocks to inclusive simulation. Insights: By sharing the perspectives of GMs in patient simulation, this study demonstrates that GMs can also benefit from engagement with medical education, as the SPs in our study described hope, empowerment, and engagement as positive aspects of participation. This study also shows that GMs' lived experiences seeking medical care were instrumental in their ability to note gaps, which provides valuable insight for other institutions attempting to improve students' GM clinical skills. Further, GM SPs' perspectives are valuable to provide a rationale and guidance to other schools implementing gender-affirming education. Efforts to create and implement gender-affirming care curriculum should include GMs in order to build partnerships and prioritize the voices and agency of GMs.

Engaging the Transgender Community to Improve Medical Education and Prioritize Healthcare Initiatives.

Phenomenon: Transgender patients experience discrimination, limited access to care, and inadequate provider knowledge in healthcare settings. Medical education to address transgender-specific disparities is lacking. Research that engages transgender community members may help address health disparities by empowering patients, increasing trust, and informing medical curricula to increase competence. APPROACH: A 2015 Community Forum on Transgender Health Care was hosted at the University of Louisville School of Medicine, which included healthcare professionals and transgender community members to facilitate dialogue among mixed-participant groups using a World Café model. Fifty-nine participants discussed the status of transgender healthcare and made recommendations for local improvements. A follow-up survey was administered to 100 individuals, including forum participants and their referrals. The forum discussion and survey responses were analyzed to determine common perceptions of transgender healthcare, priorities for improvement interventions, and themes to inform curriculum. FINDINGS: The community forum discussion showed that local transgender care is overwhelmingly underdeveloped and unresponsive to the needs of the transgender community. The follow-up survey revealed that priorities to improve transgender care included a multidisciplinary clinic for lesbian, gay, bisexual, and transgender (LGBT) patients, an LGBT-friendly network of physicians, and more training for providers and support staff. This mutually constructive engagement experience influenced reform in undergraduate curricula and continuing education opportunities. Insights: Community engagement in healthcare disparities research can cultivate improbable discussions, yield innovative insight from marginalized populations, and build relationships with community members for future collaborations and interventions. Societal acceptance of transgender identities, which could be promoted through healthcare providers, could stimulate significant progress in transgender healthcare. Supplemental educational interventions for practicing physicians will improve the current conditions of transgender healthcare, but a comprehensive medical school curriculum specifically for transgender health that includes interactions between the transgender community and medical students could be particularly impactful.

SGK1 regulation by miR-466g in cortical collecting duct cells.

Micro-RNAs (miRNAs) are noncoding RNAs that bind target mRNA transcripts and modulate gene expression. In the cortical collecting duct (CCD), aldosterone stimulates the expression of genes that increase activity of the epithelial sodium channel (ENaC); in the early phase of aldosterone induction, one such gene is serum and glucocorticoid regulated kinase 1 (SGK1). We hypothesized that aldosterone regulates the expression of miRNAs in the early phase of induction to control the expression of target genes that stimulate ENaC activity. We treated mpkCCDc14 cells with aldosterone or vehicle for 1 h and used a miRNA microarray to analyze differential miRNA expression. We identified miR-466g as a miRNA that decreased by 57% after 1 h of aldosterone treatment. Moreover, we identified a putative miR-466g binding site in the 3'-untranslated region of SGK1. We constructed an SGK1 3'-untranslated region luciferase reporter and found that cotransfection of miR-466g suppressed luciferase activity in human embryonic kidney-293 cells in a dose-dependent manner. Deletion or introduction of point mutations that disrupt the miR-466g target site attenuated miR-466g-directed suppression of luciferase activity. Finally, we generated stably transduced mpkCCDc14 cell lines overexpressing miR-466g. Cells overexpressing miR-466g demonstrated 12.9-fold lower level of SGK1 mRNA compared with control cells after 6 h of aldosterone induction; moreover, cells overexpressing miR-466g exhibited 25% decrease in amiloride-sensitive current after 6 h of aldosterone induction and complete loss of amiloride-sensitive current after 24 h of aldosterone induction. Our findings implicate miR-466g as a novel early-phase aldosterone responsive miRNA that regulates SGK1 and ENaC in CCD cells.

Barriers to USMLE Step-1 accommodations: Students with Type 1 Diabetes.

PURPOSE: Students who earn their medical doctorate (MD) in the U.S. must pass the United States Medical Licensing Exam (USMLE) Step-1. The application process for students with disabilities who seek Step-1 accommodations can be arduous, barrier-ridden, and can impose a significant burden that may have long-lasting effects. We sought to understand the experiences of medical students with Type-1 Diabetes (T1D) who applied for Step-1 accommodations. METHODS: A Qualtrics survey was administered to students enrolled in Liaison Committee on Medical Education (LCME)-accredited MD programs who disclosed having a primary diagnosis of T1D. Basic counts and qualitative inductive analyses were conducted. RESULTS: Of the 21 surveys sent, 16 (76.2%) participants responded. Of the 16 respondents, 11 (68.8%) applied for USMLE Step-1 accommodations, whereas 5 (31.2%) did not. Of the 11 who applied for accommodations, 7 (63.6%) received the accommodations requested, while 4 (36.4%) did not. Of those who received the accommodations requested, 5/7 (71.4%) experienced at least one diabetes-related barrier on exam day. Of those who did not apply for Step-1 accommodations, 4/5 (80%) participants reported experiencing at least one diabetes-related barrier on exam day. Overall, 11/16 (68.8%) students experienced barriers on exam day with or without accommodations. Qualitative analysis revealed themes among participants about their experience with the process: frustration, anger, stress, and some areas of general satisfaction. CONCLUSIONS: This study reports the perceptions of students with T1D about barriers and inequities in the Step-1 accommodations application process. Students with and without accommodations encountered T1D-related obstacles on test day.

Oxidative stress-responsive microRNA-320 regulates glycolysis in diverse biological systems.

Glycolysis is the initial step of glucose catabolism and is up-regulated in cancer cells (the Warburg Effect). Such shifts toward a glycolytic phenotype have not been explored widely in other biological systems, and the molecular mechanisms underlying the shifts remain unknown. With proteomics, we observed increased glycolysis in disused human diaphragm muscle. In disused muscle, lung cancer, and H(2)O(2)-treated myotubes, we show up-regulation of the rate-limiting glycolytic enzyme muscle-type phosphofructokinase (PFKm, >2 fold, P<0.05) and accumulation of lactate (>150%, P<0.05). Using microRNA profiling, we identify miR-320a as a regulator of PFKm expression. Reduced miR-320a levels (to ∼50% of control, P<0.05) are associated with the increased PFKm in each of these diverse systems. Manipulation of miR-320a levels both in vitro and in vivo alters PFKm and lactate levels in the expected directions. Further, miR-320a appears to regulate oxidative stress-induced PFKm expression, and reduced miR-320a allows greater induction of glycolysis in response to H(2)O(2) treatment. We show that this microRNA-mediated regulation occurs through PFKm's 3' untranslated region and that Ets proteins are involved in the regulation of PFKm via miR-320a. These findings suggest that oxidative stress-responsive microRNA-320a may regulate glycolysis broadly within nature.

Engaging Premedical Students in Medical Education Research: Benefits of Clinical Skills Observation Studies.

PURPOSE: Observations requiring evaluation and critical thinking can be powerful learning experiences. Video-recorded standardized patient encounters are underused resources for evaluation and research. The authors engaged premedical students in medical education research reviewing standardized patient encounters. This study aims to explore participant perceptions of the research experience and how they gained clinical skills. METHOD: This mixed-method study was completed between 2019 and 2022. Premedical participants coded medical students' clinical skills in video-recorded standardized patient encounters. Each participant also completed their own new patient history in a standardized patient encounter at both the beginning and end of their research project. Participants then completed an end-of-program debrief to discuss their experiences coding the clinical skills encounters. The authors coded communication skills implemented in the pre/postencounters and completed a thematic analysis of the debrief transcripts. RESULTS: All 21 participants demonstrated significant clinical skills gain after their research project, which included spending more time with the patient (pre-M=5 minutes, post-M=19 minutes, t=13.2, P<.001) and asking more questions (pre-M=13, post-M=40, t=9.3, P<.001). Prior clinical experience did not influence pre- or postoutcomes, but the number of videos coded was associated with asking more questions in the postencounter. Participants described learning actively and reflected that their clinical skills research project gave them greater insight into patient-care aspects of medical school and how medical students learn. CONCLUSIONS: These data demonstrate that observational studies in which premedical students evaluate standardized patient encounters gave the students context to medical education while enabling them to develop and transfer their own clinical skills. Studies observing standardized patient encounters provide rich insight into clinical skills development, and this work generates both research outcomes and actionable program evaluation data for medical educators. Purposefully engaging premedical students in such experiential learning opportunities benefits the students and helps cultivate early medical education pathways for these learners.

Gender Diverse Representation in Patient Simulation: A Scoping Review.

PURPOSE: Despite recent advocacy for transgender and nonbinary (TGNB) health competencies in medical education, there is little guidance on how to represent diverse gender identities for clinical skills training. Published literature is one of few resources available to inform educators' decisions, so this study aims to summarize how medical education scholarship portrays TGNB identities in patient simulation. METHOD: This scoping review used PRISMA guidelines with search strings encompassing diverse gender identities and patient simulation. This search was completed in July 2021, and all years of publication were included. The authors completed a 3-tiered review to identify relevant studies and then extracted data to summarize how TGNB patients were portrayed and training outcomes. RESULTS: After screening 194 total articles, 44 studies met the criteria for full review. Of these, 22 studies involved TGNB simulated patient cases. Within these, 15 (68%) reported the specific gender identities represented in the patient case, revealing mostly binary transgender identities. Sixteen studies (73%) reported the gender identities of all actors who portrayed the patient. The identities of all patients and actors matched in only 10 articles (45%), indicating that most programs portray TGNB identities with cisgender or unspecified standardized patients. Nearly all studies reported desirable learner outcomes. Several noted the advantage of authenticity in recruiting TGNB actors and the need to achieve more accurate representation of TGNB patients. CONCLUSIONS: Educators are increasingly representing TGNB identities in clinical skills training. These results show a lack of nonbinary representation and discrepancies between TGNB patient cases and standardized patient identities. These data also suggest that simulation programs need and desire better recruitment strategies within TGNB communities. Because TGNB communities are not a monolith, reporting out and analyzing gender identities of simulation cases and people hired to portray TGNB patients helps ensure that TGNB care is taught effectively and respectfully.

Gender-Affirming Care With Transgender and Genderqueer Patients: A Standardized Patient Case.

Introduction: Transgender and gender-diverse (TGD) patients experience health disparities and bias in health care settings. To improve care for TGD patients, medical trainees can practice gender-affirming care skills such as inclusive communication and discussing hormone therapy through patient simulation. Systematically evaluating these simulation outcomes also helps educators improve training on gender-affirming care. Methods: A standardized patient case with a patient establishing primary care was developed for rising third-year medical students. The case featured multiple patient iterations to portray individuals with the same health history but a different gender identity and/or sex assigned at birth. Each student was randomly assigned to one patient encounter. Gender-affirming care skills were assessed through standardized patient checklists, postencounter notes, and preventive care recommendations. Results: Over 2 years, 286 students participated in the simulation. Transgender men and women, cisgender men and women, and genderqueer patients were portrayed. Performance gaps such as misgendering patients and incorrect cancer screening recommendations based on perceived gender identity (rather than sex assigned at birth) were documented. Ninety-eight percent of students agreed that the encounter helped them practice clinical skills needed to see actual patients, and students described the case as challenging but important. Discussion: This case served dual roles for medical training: (1) Students working with TGD patients practiced skills for gender-affirming care, and (2) portraying TGD patients along with cisgender patients allowed educators to identify biased recommendations that necessitated additional training. The outcomes further highlighted the importance of students routinely practicing gender-inclusive communication with all patients during simulation.

LGBTQ+ Microaggressions in Health Care: Piloting an Observation Framework in a Standardized Patient Assessment.

Microaggressions are subtle derogatory behaviors that unintentionally communicate hostility toward marginalized social groups. This article describes the preliminarily validation of a framework for observing LGBTQ+ microaggressions in health care, which can lead to distrust and disengagement from the healthcare system. Coders used the framework to observe microaggressions in video-recorded clinical-skills assessments with medical students who elicited health histories from standardized patients. Microaggression classifications were reviewed to determine construct reliability and the presence/absence among eight framework categories. Among 177 encounters with sexual and gender minority standardized patients, heteronormative/cisnormative language and assumptions occurred in the largest proportion of encounters (85.3%). Only identity-based referrals decreased significantly after a clinical skills intervention (20.0% to 4.9%, p = .01). These outcomes show that LGBTQ+ healthcare microaggressions are pervasive and will likely require nuanced training to address them. This groundwork can also be used to develop scales for patients and observers to identify microaggressions and assess perceived impact.

MicroRNA-373 induces expression of genes with complementary promoter sequences.

Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.

Gender Minorities in Simulation: A Mixed Methods Study of Medical School Standardized Patient Programs in the United States and Canada.

PURPOSE: A provider's ability to translate knowledge about transgender health to affirming patient care is key to addressing disparities. However, standardized patient (SP) programs have little published guidance for gender-affirming care or addressing disparities experienced by transgender and nonbinary patients. METHOD: Between 2018 and 2019, we invited all 208 accredited US and Canadian medical schools to participate in a study to determine how gender minorities are represented in SP encounters. Responding programs (n = 59, response rate = 28%) that represented patients with diverse gender identities were invited to complete semistructured interviews about SP case content, impact, and barriers to this work. Discussions were analyzed using a modified grounded theory method. RESULTS: Fifty nine of 208 eligible programs (response rate = 28.3%) completed our survey and 24 completed interviews. More than half of programs used gender minority SPs (n = 35, 59.3%). More than half of the programs also reported portraying gender minority cases (n = 31, 52.5%). Interviewees described how effective SP simulation required purposeful case development, engaging subject matter experts with lived experience, and ensuring psychological safety of gender minority SPs. Barriers included recruitment, fear of disrespecting gender minority communities, and transphobia. Engaging gender minorities throughout case development, training, and implementation of SP encounters was perceived to reduce bias and stereotyping, but respondents unanimously desired guidance on best practices on SP methodology regarding gender identity. CONCLUSIONS: Many programs have established or are developing SP activities that portray gender minority patients. Effective SP simulation hinges on authenticity, but the decisions around case development and casting vary. Specifically, programs lack consensus about who should portray gender minority patients. This research suggests that input from gender minority communities both to inform best practices at the macro level and in an ongoing advisory capacity at the program level will be essential to teach gender-affirming care.

Double stranded-RNA-mediated activation of P21 gene induced apoptosis and cell cycle arrest in renal cell carcinoma.

Small double stranded RNAs (dsRNA) are a new class of molecules which regulate gene expression. Accumulating data suggest that some dsRNA can function as tumor suppressors. Here, we report further evidence on the potential of dsRNA mediated p21 induction. Using the human renal cell carcinoma cell line A498, we found that dsRNA targeting the p21 promoter significantly induced the expression of p21 mRNA and protein levels. As a result, dsP21 transfected cells had a significant decrease in cell viability with a concomitant G1 arrest. We also observed a significant increase in apoptosis. These findings were associated with a significant decrease in survivin mRNA and protein levels. This is the first report that demonstrates dsRNA mediated gene activation in renal cell carcinoma and suggests that forced over-expression of p21 may lead to an increase in apoptosis through a survivin dependent mechanism.

Genistein down-regulates androgen receptor by modulating HDAC6-Hsp90 chaperone function.

Androgen receptor (AR) is a ligand-activated transcription factor belonging to the steroid hormone receptor family and is very important for the development and progression of prostate cancer. The soy isoflavone genistein has been shown previously to down-regulate AR in androgen-dependent prostate cancer cell lines such as LNCaP. However, the mechanism(s) by which AR is down-regulated by genistein is still not known fully. We show a new mechanism by which genistein inhibits AR protein levels. We show that genistein-treated LNCaP cells exhibit increased ubiquitination of AR, suggesting that AR protein is down-regulated via a proteasome-mediated pathway. AR is normally stabilized by the chaperone activity of the heat shock protein Hsp90. The increased ubiquitination of AR after genistein treatment is attributed to decreased Hsp90 chaperone activity as assessed by its increased functionally inactive acetylated form. Consistent with this result, we find that HDAC6, which is a Hsp90 deacetylase, is inhibited by the antiestrogenic activity of genistein. Hence, in this study, we elucidate a novel mechanism of AR down-regulation by genistein through inhibition of HDAC6-Hsp90 cochaperone function required to stabilize AR protein. Our results suggest that genistein could be used as a potential chemopreventive agent for prostate cancers along with known inhibitors of HDAC6 and Hsp90.

HDAC3 overexpression and colon cancer cell proliferation and differentiation.

An immunohistochemical analysis of human colorectal adenocarcinomas showed that cancer cells express widely varying levels of HDAC3. The SW480 colon cancer cell line was found to express high levels of HDAC3 compared to other colon cancer cell lines. p21 was poorly induced in SW480 cells relative to the lower HDAC3-expressing HT-29 cells. RNAi-induced reduction of HDAC3 in SW480 cells increased their constitutive, butyrate-, TSA-, and TNF-alpha-induced expression of p21, but did not cause all the gene expression changes induced upon general histone deacetylase (HDAC) inhibition. SW480 cells with lower HDAC3 expression appeared to be poised for gene expression responses with increased histone H4-K12 acetylation, but not K5, K8, or K16 acetylation. Even though p21 was readily activated in HT29 cells, HDAC3 siRNA nonetheless stimulated p21 expression in these cells to a greater degree than HDAC1 and HDAC2 siRNA. SW480 cells with lower HDAC3 levels displayed an enhanced cell cycle arrest and growth inhibition by butyrate, but without changes in apoptosis or sensitivity to chemotherapeutic agents. As reported for other colon cancer cell lines, butyrate induced the rapid downregulation of the secretory cell differentiation markers mucin 2 and intestinal trefoil factor in SW480 cells. Interestingly, selective HDAC3 inhibition was sufficient to downregulate these genes. Our data support a central role for HDAC3 in regulating the cell proliferation and differentiation of colon cancer cells and suggest a potential mechanism by which colon cancers may become resistant to luminal butyrate.

Hsp70B' regulation and function.

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B'. Flow cytometry was used to assay hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B' and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.

Hsp70B' regulation and function.

Heat shock protein (Hsp) 70B' is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B' and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of Hsp70B'. Flow cytometry was used to assay Hsp70B' promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B'- and Hsp72-sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B' is a secondary responder. Interestingly ZnSO4 induces Hsp70B' and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B'. Both Hsp70B' and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B' contributes to cell survival.

Circumvention and reactivation of the p53 oncogene checkpoint in mouse colon tumors.

The p53 tumor suppressor protein is sequence-normal in azoxymethane (AOM)-induced mouse colon tumors, making them a good model for human colon cancers that retain a wild type p53 gene. Cellular localization and co-immunoprecipitation experiments using a cell line derived from an AOM-induced colon tumor (AJ02-NM(0) cells) pointed to constitutively expressed Mdm2 as being an important negative regulator of p53 in these cells. Although the Mdm2 inhibitory protein p19/ARF was expressed in AJ02-NM(0) cells, its level of expression was not sufficient for p53 activation. We tested the response of AJ02-NM(0) cells to the recently developed Mdm2 inhibitor, Nutlin-3. Nutlin-3 was found to activate p53 DNA binding in AJ02-NM(0) cells, to a level comparable to doxorubicin and 5-fluorouracil (5-FU). In addition, Nutlin-3 increased expression of the p53 target genes Bax and PERP to a greater extent than doxorubicin or 5-FU, and triggered a G2/M phase arrest in these cells, compared to a G1 arrest triggered by doxorubicin and 5-FU. The differences in the cellular response may be related to differences in the kinetics of p53 activation and/or its post-translational modification status. In an ex vivo experiment, Nutlin-3 was found to activate p53 target gene expression and apoptosis in AOM-induced tumor tissue, but not in normal adjacent mucosa. Our data indicate that Mdm2 inhibitors may be an effective means of selectively targeting colon cancers that retain a sequence-normal p53 gene while sparing normal tissue and that the AOM model is an appropriate model for the preclinical development of these drugs.

HDACs and the senescent phenotype of WI-38 cells.

BACKGROUND: Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells. RESULTS: Pre- and post-senescent WI-38 cells were treated with the HDAC inhibitors butyrate or trichostatin A (TSA). Following HDAC inhibitor treatment, pre-senescent cells increased p21WAF1 and beta-galactosidase expression, assumed a flattened senescence-associated morphology, and maintained a lower level of proteasome activity. These alterations also occurred during normal replicative senescence of WI-38 cells, but were not accentuated further by HDAC inhibitors. We also found that HDAC1 levels decline during normal replicative senescence. CONCLUSION: Our findings indicate that HDACs impact numerous phenotypic changes associated with cellular senescence. Reduced HDAC1 expression levels in senescent cells may be an important event in mediating the transition to a senescent phenotype.