Abstract Previously published gene expression analyses suggested that apoptotic function may be reduced in humans relative to chimpanzees and led to the hypothesis that this difference may contribute to the relatively larger size of the human brain and the increased propensity of humans to develop cancer. In this study, we sought to further test the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees by conducting a series of apoptotic function assays on human, chimpanzee and macaque primary fibroblastic cells. Human cells consistently displayed significantly reduced apoptotic function relative to the chimpanzee and macaque cells. These results are consistent with earlier findings indicating that apoptotic function is reduced in humans relative to chimpanzees.

Citation: Arora G, Mezencev R, McDonald JF (2012) Human Cells Display Reduced Apoptotic Function Relative to Chimpanzee Cells. PLoS ONE 7(9): e46182. https://doi.org/10.1371/journal.pone.0046182 Editor: Christopher Mark Norris, University of Kentucky, United States of America Received: February 23, 2012; Accepted: August 29, 2012; Published: September 28, 2012 Copyright: © Arora et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was carried out with the financial support of the Georgia Tech Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Although the human and chimpanzee genomes are highly similar (>98.5% sequence identity) [1], the two species have accumulated significant differences in a number of phenotypic traits since diverging from a common ancestor 6–8 million years ago [2]. Among the most notable of these phenotypic differences is the substantially larger size of the human brain (∼3×) and associated areas of specialized function [3], [4]. A second notable difference between the two species is the significantly higher incidence of cancer in humans relative to chimpanzee (e.g., [5]–[8]) even after adjustment for differences in lifespan [9]. We recently proposed that the evolutionary differences in these traits may, at least in part, be commonly rooted in molecular differences in apoptosis, or programmed cell death [10]. Neurons in the human cerebral cortex are only produced during early development [11]. As neurons in the neocortex are lost over the life span of humans and other primates, they are not replaced. Thus, one potential mechanism by which brain size could have been increased in humans relative to the other primates is by a reduction in the rate of programed cell death. In support of this model, we previously observed that apoptotic pathway genes are differentially expressed between human and chimpanzee brains and other tissues consistent with a generally reduced apoptotic function in humans [10]. Since reduced apoptotic function is well known to be associated with an increased propensity for cancer [5]–[8], we hypothesized that selection for increased cognitive function in humans may have coincidently contributed to an increased propensity for cancer [10]. A key premise of this hypothesis is the validity of the assertion that apoptotic function is indeed reduced in humans relative to chimpanzees. In an initial effort to experimentally test this hypothesis, we conducted a series of experiments designed to detect differences in apoptotic function among human, chimpanzee and macaque (out-group) primary fibroblast cells. The results are uniformly consistent with the hypothesis that apoptotic function is significantly reduced in humans relative to chimpanzees and macaques.

Discussion The fact that the apoptotic process plays a key role in both brain development and cancer progression, led us to previously hypothesize that the differences in brain size and propensity for cancer that exists between humans and chimpanzees may both be associated, at least in part, with differences in apoptotic function [10]. In agreement with this hypothesis, we previously observed that differences in the expression of apoptotic pathway genes between human and chimpanzee brains and other tissues were consistent with reduced apoptotic function in humans [10]. In this study, we sought to further test the hypothesis that apoptotic function is reduced in humans relative to chimpanzees by monitoring the relative response of human and chimpanzee primary fibroblast cells to apoptotic-inducing agents. A variety of assays were conducted using 6 independently established primate primary cell cultures (2 human, 2 chimpanzee and 2 macaque) and the results were uniformly consistent with the hypothesis that the apoptotic function in humans is significantly reduced relative to chimpanzees and macaques. At low concentrations of the apoptosis-inducing agent MMC (1–10 µM and 15 µM), chimpanzee cells displayed a significantly lower number of viable cells compared to the human samples. The nuclear morphology of the chimpanzee cells at these concentrations was typical of apoptosis showing nuclear condensation and fragmentation. In contrast, the nuclear morphology of the human cells at these low concentrations was similar to untreated cells (Figure 3A), indicating that very little or no apoptosis was occurring in the human samples. Comparisons of the IC 50 values of MMC treated human and chimpanzee cells confirmed that the chimpanzee cells were more sensitive to MMC-induced apoptosis. Although further testing will be required before definitive conclusions can be drawn, our results are consistent with the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees. To determine whether this putative difference in apoptotic function between humans and chimpanzees is most likely to have occurred in the human or chimpanzee lineage, we used macaque as an out-group in our assays. The results consistently indicated that macaque cells behaved similarly to chimpanzee cells in displaying higher sensitivity to apoptosis-inducing agents relative to human cells. These findings suggest that the reduced apoptotic function associated with human cells is an evolutionarily derived condition occurring within the human lineage subsequent to the divergence of humans and chimps from a common ancestor ∼6 MYA. Consistent with this view are previous findings indicating that apoptotic genes display accelerated rates of evolutionary change within the human lineage relative to the other primates [27].

Materials and Methods Fibroblast Cells Primary human, chimpanzee and macaque skin fibroblast cells were obtained from Coriell cell repositories (Camden, NJ, USA). Human cells: AG13153 – established from a 30-year-old male; AG07307 – established from a 40-year-old female; Chimpanzee cells: S006007 – established from a 22-year-old male; S005795 – established from a 26-year-old female; Macaque cells: AG07915 - established from a 12-year-old male; AG07128 – established from an 11-year-old female. Cell Culture Human primary fibroblasts were cultured in Minimum Essential Medium with Hank's BSS supplemented with 2 mM L-glutamine (Mediatech, VA, USA), antibiotic/antimyotic (100 IU/ml Penicillin, 100 µg/ml Streptomycin, 0.25 µg/ml Amphotericin B, Mediatech, VA, USA), 26 mM Hepes (Sigma, MO, USA) and 10% FBS (Gibco, NY, USA) at 37°C in humidified air with 5% CO 2 . Chimpanzee primary fibroblasts were cultured in Minimum Essential Medium alpha-modification with nucleosides with 2 mM L-glutamine (Mediatech, VA, USA), antibiotic/antimyotic (100 IU/ml Penicillin, 100 µg/ml Streptomycin, 0.25 µg/ml Amphotericin B, Mediatech, VA, USA) and 10% FBS (Gibco, NY, USA) at 37°C in humidified air with 5% CO 2 . Macaque primary fibroblasts were cultured in Minimum Essential Medium alpha-modification with nucleosides with 2 mM L-glutamine (Mediatech, VA, USA), antibiotic/antimyotic (100 IU/ml Penicillin, 100 µg/ml Streptomycin, 0.25 µg/ml Amphotericin B, Mediatech, VA, USA) and 15% FBS (Gibco, NY, USA) at 37°C in humidified air with 5% CO 2 . The growth rates for each of the cells were determined in 96 well plates using the TOX-8 (Sigma-Aldrich, St. Louis, MO, USA) cell cytotoxicity assay. Passage numbers at which tests were done were as follows: human (AG13153) P14–P16; chimpanzee (S006007) P14–P16; macaque (AG07915) P16–P18; human (AG07307) P17–P19; chimpanzee (S005795) P18–P20; and macaque (AG07128) P18–P20. Cell Viability Assay Mitomycin C was obtained from Sigma Aldrich, MO, USA, and staurosporine from Fisher Scientific, PA, USA. The cell viability experiments were conducted in 96 well plates using the resazurin-based In Vitro Toxicology Assay Kit TOX-8 (Sigma-Aldrich, St. Louis, MO, USA) as previously described [28]. For each cell type, the number of cells seeded per well (as determined by pre-test optimization) was 80,000 cells/ml for the human cells, 120,000 cells/ml for the chimpanzee cells and 60,000 cells/ml for the macaque cells. The cells were treated over a range of concentrations of staurosporine and MMC for 48 and 72 hours respectively in RPMI growth medium supplemented with 5% FBS and penicillin (100 IU/mL), streptomycin (100 µg/mL) and amphotericin B (0.25 µg/mL). Hoechst Staining The cells in the 96 well plates treated with MMC for 72 hours and untreated control cells were washed twice with 100 µl of PBS, fixed with 10% buffered formal-saline for 30 minutes and stained with 10 µg/ml of Hoechst 33342 (Sigma-Aldrich) in H 2 O for 15 minutes. The cells were then visualized using a fluorescence microscope (Olympus IX51, Olympus, NJ, USA) and photographed using an Olympus DP72 digital camera. Caspase-3/7 Activity Assay The human (AG13153) and chimpanzee (S006007) cells were allowed to grow for 24 hours in white walled cell culture-treated 96 well plates, and with staurosporine for 48 hours to induce apoptosis. Following treatment with the drug, 100 µl of the Caspase-3/7 Glo reagent (Promega Corporation, WI, USA) was added, followed by incubation at room temperature for 30 minutes to generate a luminescence signal. The caspase-3/7 activities were determined by measuring luminescence signal using a microplate reader (Spectramax Gemini XS, Molecular Devices, CA, USA). Measurement of Mitochondrial Transmembrane Potential Changes in ΔΨ m upon treatment with MMC were detected by flow cytometry experiment using tetramethylrhodamine ethyl ester (TMRE). Human (AG07307) and chimpanzee (S005795) cells were grown in 100 mm Petri dishes for 24 hours and subsequently treated with different concentrations of MMC for 72 hours. Thereafter, adherent cells were harvested by trypsinization, combined with free-floating cells in growth medium and stained in dark with 100 nM of TMRE for 30 minutes at 37°C. TMRE fluorescence was measured using the PE channel of the BD LSR II flow cytometer (BD Biosciences, NJ, USA). Analysis of the data was carried out using FlowJo 7.6 software (Tree Star, Inc., Ashland, OR, USA). Statistical Analysis When not specified otherwise, significance of differences between means was tested by two-tailed Student's t-test. Differences in proportions of cells with dissipated ΔΨ m between MMC-treated cells were tested using two-proportion z-test. IC 50 values were determined by non-linear regression of log-transformed data using a normalized response-variable slope model (GraphPad Prism 5.01; GraphPad Software, Inc.).

Supporting Information File S1. Differential expression between human and bonobo fibroblasts of genes previously shown to be associated with resistance to MMC. https://doi.org/10.1371/journal.pone.0046182.s001 (DOCX)

Author Contributions Conceived and designed the experiments: JFM RM GA. Performed the experiments: RM GA. Analyzed the data: RM JFM GA. Contributed reagents/materials/analysis tools: JFM. Wrote the paper: JFM RM GA.