Tumor Suppression In Humans Research Articles

Abstract 3025: Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis

Abstract TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations. Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3025.

GLS2 Is a Tumor Suppressor and a Regulator of Ferroptosis in Hepatocellular Carcinoma.

This study demonstrates that the key regulator of glutaminolysis, GLS2, can limit HCC in vivo by promoting ferroptosis through αKG-dependent lipid ROS, which in turn might lay the foundation for a novel therapeutic approach.

Regulation of p53 Protein Function in Response to Heat Shock

p53 protein encoded by the TP53 gene performs one of the most important functions in the process of human tumor suppression. Mutations in the DNA-binding domain of p53 change its conformation, which contributes to the formation of aberrant intracellular protein complexes including heat shock proteins (Hsp70). This can provoke an occurrence of the aggressive types of tumors including breast cancer. Thereby, the study of the regulation mechanisms of mutant p53 in these stable complexes seems extremely relevant. The aim of this work was to study the regulation of p53 protein mutant for R175H (mutp53−R175H) under heat stress in MDA–MB-231 breast cancer cells in vitro. It was found that heat shock caused a drastic decrease in the level of wtp53 (wild-type p53) and mutp53–R175H proteins. It was gradually restored after the stress ceased. We also have found that mutp53–R175H increases the intracellular level of Hsp70 in normal conditions and reduces it after heat shock. At the same time, mutp53–R175H protein changes its intracellular localization, both in normal conditions and in response to heat shock being in the composition of Hsp70 protein complexes. Thus, the behavior of wtp53 and mutp53–R175H in response to heat shock appears to differ due to different interactions with protein complexes that regulate their stability and intracellular localization.

Generation of an Endogenous Dominant Negative Allele of chd5 Using a Novel Homologous Recombination Strategy to Reveal the Role of chd5 in Tumor Suppression and Neural Differentiation

CHD5 is a vertebrate specific member of a family of ATP‐dependent chromatin remodeling proteins. Chromodomain/Helicase/DNA‐binding (CHD) proteins play a variety of roles in vertebrate development, and loss of CHD5 causes failure of proper expression in a cohort of genes involved in neural development. CHD5 plays a critical role tumor suppression in humans, and loss of CHD5 contributes to the formation or progression of numerous cancers, in particular neuroblastoma. To understand how CHD5 plays a role in tumor suppression, it is necessary to understand how CHD5 contributes to normal development and how its normal functions are misregulated during tumor progression. Our lab is establishing zebrafish as model system in which to study the role of chd5 dependent processes.We have engineered chd5 knockout fish using CRISPR‐Cas9 technology. The resulting fish do not exhibit any overt developmental phenotypes, and do not produce tumors during adulthood (N=165, aged >16 months). We hypothesize that the closely related remodelers CHD3/4 function redundantly with CHD5, and propose to use a novel knock‐in approach to generate zebrafish carrying a dominant negative mutation in the ATPase domain that has previously been shown to result in strong mutant phenotypes when generated in closely related remodelers.To engineer a dominant negative allele of chd5 we have engineered a homologous recombination (HR) strategy that uses piggyBac transposase technology, which has previously been used in Drosophila, mice, and human cells to engineer scarless genomic modifications. This design uses two‐steps to first, introduce a homologous donor carrying the mutation of interest and a fluorescent marker for easy identification. Second, the HR positive fish will be bred with piggyBac expressing fish lines to seamlessly excise the exogenous sequences, thus leaving only the desired genome modification.Once we have engineered this method into the fish, we will be able to use piggyBac to control when and where we express the dominant negative allele of chd5 so that we can score the resulting fish for developmental and tumor phenotypes. The screening of the dominant negative, knock‐in lines is in progress. The resulting dominant negative lines will give great insight into the role of chd5 in neural development and tumor suppression.Support or Funding InformationFunding made available by: Purdue University Center for Cancer Research, Purdue University Department of Biochemistry, Purdue University College of AgricultureThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel nuclear translocation of inositol polyphosphate 4-phosphatase is associated with cell cycle, proliferation and survival

Inositol polyphosphate 4 phosphatase type I enzyme (INPP4A) has a well-documented function in the cytoplasm where it terminates the phosphatidylinositol 3-kinase (PI 3-K) pathway by acting as a negative regulator. In this study, we demonstrate for the first time that INPP4A shuttles between the cytoplasm and the nucleus. Nuclear INPP4A is enzymatically active and in dynamic equilibrium between the nucleus and cytoplasm depending on the cell cycle stage, with highest amounts detected in the nucleus during the G0/G1 phase. Moreover, nuclear INPP4A is found to have direct proliferation suppressive activity. Cells constitutively overexpressing nuclear INPP4A exhibit massive apoptosis. In human tissues as well as cell lines, lower nuclear localization of INPP4A correlate with cancerous growth. Together, our findings suggest that nuclear compartmentalization of INPP4A may be a mechanism to regulate cell cycle progression, proliferation and apoptosis. Our results imply a role for nuclear-localized INPP4A in tumor suppression in humans.

Reversine Induced Multinucleated Cells, Cell Apoptosis and Autophagy in Human Non-Small Cell Lung Cancer Cells.

Reversine, an A3 adenosine receptor antagonist, has been shown to induce differentiated myogenic-lineage committed cells to become multipotent mesenchymal progenitor cells. We and others have reported that reversine has an effect on human tumor suppression. This study revealed anti-tumor effects of reversine on proliferation, apoptosis and autophagy induction in human non-small cell lung cancer cells. Treatment of these cells with reversine suppressed cell growth in a time- and dosage-dependent manner. Moreover, polyploidy occurred after reversine treatment. In addition, caspase-dependent apoptosis and activation of autophagy by reversine in a dosage-dependent manner were also observed. We demonstrated in this study that reversine contributes to growth inhibition, apoptosis and autophagy induction in human lung cancer cells. Therefore, reversine used as a potential therapeutic agent for human lung cancer is worthy of further investigation.

2P102 ヒト癌抑制遺伝子候補101F6によるカスパーゼ非依存性細胞死の機構分析(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

2P102 ヒト癌抑制遺伝子候補101F6によるカスパーゼ非依存性細胞死の機構分析(03. 膜蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

P53 integrates host defense and cell fate during bacterial pneumonia

Cancer and infection are predominant causes of human mortality and derive, respectively, from inadequate genomic and host defenses against environmental agents. The transcription factor p53 plays a central role in human tumor suppression. Despite its expression in immune cells and broad responsiveness to stressors, it is virtually unknown whether p53 regulates host defense against infection. We report that the lungs of naive p53(-/-) mice display genome-wide induction of NF-κB response element-enriched proinflammatory genes, suggestive of type 1 immune priming. p53-null and p53 inhibitor-treated mice clear Gram-negative and -positive bacteria more effectively than controls after intrapulmonary infection. This is caused, at least in part, by cytokines produced by an expanded population of apoptosis-resistant, TLR-hyperresponsive alveolar macrophages that enhance airway neutrophilia. p53(-/-) neutrophils, in turn, display heightened phagocytosis, Nox-dependent oxidant generation, degranulation, and bacterial killing. p53 inhibition boosts bacterial killing by mouse neutrophils and oxidant generation by human neutrophils. Despite enhanced bacterial clearance, infected p53(-/-) mice suffer increased mortality associated with aggravated lung injury. p53 thus modulates host defense through regulating microbicidal function and fate of phagocytes, revealing a fundamental link between defense of genome and host during environmental insult.

Abstract 2034: The Fbw7 tumor suppressor: Genetic and biochemical approaches to study a dimeric ubiquitin ligase

Abstract Fbw7, the human ortholog of yeast Cdc4, is the substrate-recognition component of a cullin-RING ubiquitin ligase and regulates a number of oncoprotein substrates including cyclin E, c-Myc, Notch, and c-Jun. A growing number of additional pro-growth and pro-survival factors have been more recently described as Fbw7 targets. Accordingly, Fbw7 is a tumor suppressor and is mutated in approximately ten percent of human malignancies. Its loss leads to chromosomal instability and tumorigenesis. Fbw7 binds its substrates after they become phosphorylated within a consensus sequence called a Cdc4 phospho-degron (CPD). CPD phosphorylation creates a high-affinity binding site for Fbw7, which brings the ligase machinery into proximity with the substrate, leading to ubiquitylation and proteasomal degradation of the substrate. Interestingly, Fbw7 contains a conserved dimerization domain and we can show the protein primarily forms dimers in cells. One Fbw7 dimer contains two substrate-binding interfaces and may make two independent CPD contacts, but the physiologic function of Fbw7 dimerization is still elusive. Using gene-targeting, we studied the effects of ablating Fbw7 dimerization in vivo and found endogenous cyclin E and c-Myc regulation are moderately affected. Using purified Fbw7 monomers or dimers in in vitro ubiquitylation assays, we observed that a dimeric ligase is able to conjugate ubiquitin moieties onto a wider variety of substrate lysine residues than can the monomeric ligase. This is likely due to the greater spatial reach of the dimer. Additionally, preliminary data shows that the dimer may be capable of catalyzing the formation of non-K48-linked polyubiquitin chains, whereas the monomer is not. Cancer-associated mutations in Fbw7 most often target three arginine residues that form a pocket for binding phosphorylated substrate CPDs. Mutation of any of these arginines render Fbw7 unable to bind substrates and properly regulate their abundance. However, these mutations occur typically in only one allele. Dimerization of Fbw7 may explain why such heterozygous mutations are sufficient for tumorigenesis: the mutant form may dominantly inhibit enzyme function of the wild-type allele through dimerization. We have generated hetero-typic Fbw7 dimers containing one wild-type and one mutant protein in each dimer and are currently studying its binding and ubiquitylation capabilities. Given the importance of Fbw7 and its substrates in tumor-progression pathways, the results of this study will further our understanding of human tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2034. doi:1538-7445.AM2012-2034

Tumor-suppressing Function of Caspase-2 Requires Catalytic Site Cys-320 and Site Ser-139 in Mice

The multifunctional caspase-2 protein is involved in apoptosis, NF-κB regulation, and tumor suppression in mice. However, the mechanisms of caspase-2 responsible for tumor suppression remain unclear. Here we identified two sites of caspase-2, the catalytic Cys-320 site and the Ser-139 site, to be important for suppression of cellular transformation and tumorigenesis. Using SV40- and K-Ras-transformed caspase-2 KO mouse embryonic fibroblast cells reconstituted with expression of wild-type, catalytic dead (C320A), or Ser-139 (S139A) mutant caspase-2, we demonstrated that similar to caspase-2 deficiency, when Cys-320 and Ser-139 were mutated, caspase-2 lost its ability to inhibit cellular transformation and tumorigenesis. These mutant cells exhibited enhanced cell proliferation, elevated clonogenic activity, accelerated anchorage-independent growth, and transformation and were highly tumorigenic, rapidly producing large tumors in athymic nude mice. Investigation into the underlying mechanism showed that these two residues are needed for caspase-2 to suppress NF-κB activity, promote apoptosis, and sustain the G(2)/M checkpoint following DNA damage induction. In addition, tumors in nude mice derived from the two mutant cell lines had higher constitutive NF-κB activity and elevated expression of NF-κB targets of antiapoptotic proteins Bcl-xL, XIAP, and cIAP2. A reduction in caspase-2 mRNA was associated with multiple types of cancers in patients. Together, these observations suggest the combined functions of caspase-2 in suppressing NF-κB activation, promoting apoptosis, and sustaining G(2)/M checkpoint contribute to caspase-2 tumor-suppressing function and that caspase-2 may also impact tumor suppression in humans. These findings provide insight into tumor suppression at the cross-roads of apoptosis, cell cycle checkpoint, and NF-κB pathways.

Noise Amplification in Human Tumor Suppression following Gamma Irradiation

The influence of noise on oscillatory motion is a subject of permanent interest, both for fundamental and practical reasons. Cells respond properly to external stimuli by using noisy systems. We have clarified the effect of intrinsic noise on the dynamics in the human cancer cells following gamma irradiation. It is shown that the large amplification and increasing mutual information with delay are due to coherence resonance. Furthermore, frequency domain analysis is used to study the mechanisms.

Autophagy is involved in anticancer effects of matrine on SGC-7901 human gastric cancer cells

Matrine has a wide range of pharmacological effects including antitumor activity in vitro and in vivo. Autophagy is closely associated with tumors and plays an important role in human tumor suppression, so inducing autophagy is a potential therapeutic strategy in adjuvant chemotherapy. The aim of this study was to investigate whether or not autophagy is involved in antitumor effects of matrine on human gastric cancer SGC-7901 cells, and to further elucidate the underlying molecular mechanisms. Sulphorhodamine B (SRB) assay was used to examine matrine's cytotoxicity against SGC-7901 gastric cancer cells. The effects of matrine on the cell cycle and apoptosis were measured by flow cytometry, and cellular morphology was observed under an inverted phase contrast microscope and transmission electron microscope. Monodansylcadaverine (MDC) staining was used to detect autophagy. The expression levels of Bax and Beclin 1 in SGC-7901 cells were monitored by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that matrine significantly inhibited the proliferation of SGC-7901 gastric cancer cells and induced G1-phase cell cycle arrest. Furthermore, both autophagy and apoptosis were activated during the matrine-induced death of SGC-7901 cells. Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanisms of matrine may be associated with its up-regulation of Bax expression. These findings indicate that matrine is a potent antitumor agent for treating gastric cancer. The ability of matrine to induce autophagy underlines its potential utility as a new gastric cancer treatment modality.

Enhancement of RAD51 recombinase activity by the tumor suppressor PALB2

Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a co-operative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51 and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.

Centrobin regulates the assembly of functional mitotic spindles

The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule-kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.

Caspase 2-mediated tumor suppression involves survivin gene silencing

One of the pivotal functions of endogenous tumor suppression is to oppose aberrant cell survival, but the molecular requirements of this process are not completely understood. Here, we show that caspase 2, a death effector with largely unknown functions, represses transcription of the survivin gene, a general regulator of cell division and cytoprotection in tumors. This pathway involves caspase 2 proteolytic cleavage of the NFκB activator, RIP1. In turn, loss of RIP1 abolishes transcription of NFκB target genes, including survivin, resulting in deregulated mitotic transitions, enhanced apoptosis, and suppression of tumorigenicity, in vivo. Therefore, caspase 2 functions as an endogenous inhibitor of NFκB-dependent cell survival, and this mechanism may contribute to tumor suppression in humans.

Stochastic modeling and simulation of the p53-MDM2/MDMX loop.

The p53 gene is crucial for effective tumor suppression in humans as supported by its universal inactivation in cancer cells either through mutations affecting the p53 locus directly or through aberration of its normal regulation. The p53 tumor repressor is regulated through a negative feedback loop involving its transcriptional target MDM2. MDMX is also an essential negative regulator of p53. Several computational models have been proposed to simulate the dynamics of the p53-MDM2 loop, but they do not include MDMX, only account for some basic interactions between p53 and MDM2 and cannot capture the intrinsic noise in the loop. In this article, we present a comprehensive model for the p53-MDM2/MDMX loop that accounts for most known interactions among p53, MDM2 and MDMX. Our model is characterized by a set of molecular reactions, which enables us to employ stochastic simulation to investigate the dynamics of the loop. In agreement with experiments, our results show that p53 and MDM2 undergo oscillations after DNA damage in the presence of noise, and the variation in oscillation amplitudes is much higher than that in oscillation periods. Our simulations predict that intrinsic noise contributes to 60%-70% of the total variation in oscillation amplitudes and periods. The protein levels of p53, MDM2, and MDMX after treatment with Nutlin in our simulations are also consistent with experimental results. Our simulation results further predict that p53 levels increase dramatically after MDM2 is knocked out, but increase with a much less amount after MDMX is knocked out. This may partially explain why MDM2-null and MDMX-null mouse embryos die in different developmental stages. Our stochastic model and simulation provide insights into the variability of the behavior of the p53 pathway and can be used to predict the dynamics of the pathway after certain interventions.

Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Notch1 is a p53 target gene involved in human keratinocyte tumor suppression through negative regulation of ROCK1/2 and MRCKα kinases

Little is known about the regulation and function of the Notch1 gene in negative control of human tumors. Here we show that Notch1 gene expression and activity are substantially down-modulated in keratinocyte cancer cell lines and tumors, with expression of this gene being under p53 control in these cells. Genetic suppression of Notch signaling in primary human keratinocytes is sufficient, together with activated ras, to cause aggressive squamous cell carcinoma formation. Similar tumor-promoting effects are also caused by in vivo treatment of mice, grafted with keratinocytes expressing oncogenic ras alone, with a pharmacological inhibitor of endogenous Notch signaling. These effects are linked with a lesser commitment of keratinocytes to differentiation, an expansion of stem cell populations, and a mechanism involving up-regulation of ROCK1/2 and MRCKalpha kinases, two key effectors of small Rho GTPases previously implicated in neoplastic progression. Thus, the Notch1 gene is a p53 target with a role in human tumor suppression through negative regulation of Rho effectors.

Inactivation of myopodin expression associated with prostate cancer relapse

Objectives Myopodin has recently been characterized as a tumor suppressor gene whose encoded product inhibits prostate cancer growth and metastasis both in vitro and in animal models. However, the clinical evidence of tumor suppression in humans is still lacking. In this study, we conducted a large-scale analysis of myopodin expression in prostate cancer samples to examine the relationship between myopodin expression and prostate cancer grade and stage and the probability of clinical relapse. Methods Immunostaining using anti-myopodin antibodies was performed on 746 formalin-fixed paraffin-embedded tissue samples to evaluate the level of myopodin expression in normal and malignant prostatic tissue. Results The expression of myopodin was semiquantitatively determined by immunostaining. The myopodin expression levels were examined in relation to prostate cancer grade and stage, preoperative prostate-specific antigen level, surgical margin involvement, tumor volume, and clinical relapse. The mean myopodin expression score (range 0 to 3) was 2.1 and 0.88 for benign prostatic tissue and prostate cancer, respectively. Minimal variation in myopodin expression was observed among the various grades, stages, tumor volumes, and preoperative prostate-specific antigen levels of prostate cancer. However, samples with positive surgical margins were associated with lower myopodin expression. Complete inactivation of myopodin expression correlated with a greater than 86% rate of clinical relapse. Conclusions Our results suggest that myopodin is an important predictor of prostate cancer metastasis, independent of Gleason score, preoperative prostate-specific antigen level, and tumor stage.

Cellular Aging and Transformation Suppression: A Role for Retinoic Acid Receptor β2

Cellular senescence is characterized by a finite proliferative capacity in vitro . Moreover, the proliferative capacity of dermal fibroblasts harvested from humans is inversely proportional to the age of the donor, suggesting that senescence in culture is a manifestation, at the cellular level, of processes that occur during in vivo human aging. As cellular senescence is a program that ultimately decreases cell proliferation, it has been hypothesized that the genetic mechanisms responsible for the negative growth regulation of senescence many also be involved in the suppression of neoplastic transformation. Retinoic acid (RA) and its derivatives are effective negative growth regulators and are known to inhibit tumor growth, in vitro and in vivo. As a first step in examining a role for retinoic acid in the regulation of cellular aging in human fibroblasts, we examined the expression of the nuclear receptors for RA (RARα, RARβ, and RARγ) in human donors of different ages. These studies demonstrate a selective up-regulation of RARβ, in response to RA, in fibroblasts that manifest a decreased proliferative capacity. We extend these observations to show that this finding is independent of the age of the donor and correlates with the proliferative capacity of the culture as a whole. Nuclear run-on studies show that the increase in RARβ mRNA accumulation is mediated by a striking increase in the transcription of the RARβ2 isoform. Senescent fibroblasts manifesting the transcriptional increase of the RARβ2 isoform also demonstrate transcriptional repression of the protooncogene, c-fos. Functional studies demonstrate that RARβ2, like the tumor suppressor gene p53, can inhibit oncogene-induced focus formation. These data provide further support for the contention that genetic events important in cellular senescence may also play a significant role in tumor suppression in humans Moreover, these observations suggest that RA, through transcriptional regulation of RARβ2, may mediate aspects of the negative growth control that characterizes both states.