Normal Human Diploid Fibroblasts Research Articles

Senescence-Associated Exosome Release from Human Prostate Cancer Cells

Males of advanced age represent a rapidly growing population at risk for prostate cancer. In the contemporary setting of earlier detection, a majority of prostate carcinomas are still clinically localized and often treated using radiation therapy. Our recent studies have shown that premature cellular senescence, rather than apoptosis, accounts for most of the clonogenic death induced by clinically relevant doses of irradiation in prostate cancer cells. We show here that this treatment-induced senescence was associated with a significantly increased release of exosome-like microvesicles. In premature senescence, this novel secretory phenotype was dependent on the activation of p53. In addition, the release of exosome-like microvesicles also increased during proliferative senescence in normal human diploid fibroblasts. These data support the hypothesis that senescence, initiated either by telomere attrition (e.g., aging) or DNA damage (e.g., radiotherapy), may induce a p53-dependent increase in the biogenesis of exosome-like vesicles. Ultrastructural analysis and RNA interference-mediated knockdown of Tsg101 provided significant evidence that the additional exosomes released by prematurely senescent prostate cancer cells were principally derived from multivesicular endosomes. Moreover, these exosomes were enriched in B7-H3 protein, a recently identified diagnostic marker for prostate cancer, and an abundance of what has recently been termed "exosomal shuttle RNA." Our findings are consistent with the proposal that exosomes can transfer cargos, with both immunoregulatory potential and genetic information, between cells through a novel mechanism that may be recruited to increase exosome release during accelerated and replicative cellular senescence.

Efficient Quiescent Infection of Normal Human Diploid Fibroblasts with Wild-Type Herpes Simplex Virus Type 1

Quiescent infection of cultured cells with herpes simplex virus type 1 (HSV-1) provides an important, amenable means of studying the molecular mechanics of a nonproductive state that mimics key aspects of in vivo latency. To date, establishing high-multiplicity nonproductive infection of human cells with wild-type HSV-1 has proven challenging. Here, we describe simple culture conditions that established a cell state in normal human diploid fibroblasts that supported efficient quiescent infection using wild-type virus and exhibited many important properties of the in vivo latent state. Despite the efficient production of immediate early (IE) proteins ICP4 and ICP22, the latter remained unprocessed, and viral late gene products were only transiently and inefficiently produced. This low level of virus activity in cultures was rapidly suppressed as the nonproductive state was established. Entry into quiescence was associated with inefficient production of the viral trans-activating protein ICP0, and the accumulation of enlarged nuclear PML structures normally dispersed during productive infection. Lytic replication was rapidly and efficiently restored by exogenous expression of HSV-1 ICP0. These findings are in agreement with previous models in which quiescence was established with HSV mutants disrupted in their expression of IE gene products that included ICP0 and, importantly, provide a means to study cellular mechanisms that repress wild-type viral functions to prevent productive replication. We discuss this model in relation to existing systems and its potential as a simple tool to study the molecular mechanisms of quiescent infection in human cells using wild-type HSV-1.

Genomic and proteomic profiling of oxidative stress response in human diploid fibroblasts

A number of lines of evidence suggest that senescence of normal human diploid fibroblasts (HDFs) in culture is relevant to the process of aging in vivo. Using normal human skin diploid fibroblasts, we examine the changes in genes and proteins following treatment with a mild dose of H2O2, which induces premature senescence. Multidimensional Protein Identification Technology (MudPIT) in combination with mass spectrometry analyses of whole cell lysates from HDFs detected 65 proteins in control group, 48 proteins in H2O2-treated cells and 109 proteins common in both groups. In contrast, cDNA microarray analyses show 173 genes up-regulated and 179 genes down-regulated upon H2O2 treatment. Both MudPIT and cDNA microarray analyses indicate that H2O2 treatment caused elevated levels of thioredoxin reductase 1. Semi-quantitative RT-PCR and Western-blot were able to verify the finding. Out of a large number of genes or proteins detected, only a small fraction shows the overlap between the outcomes of microarray versus proteomics. The low overlap suggests the importance of considering proteins instead of transcripts when investigating the gene expression profile altered by oxidative stress.

Oncogene-Induced Senescence Relayed by an Interleukin-Dependent Inflammatory Network

Oncogene-induced cellular senescence (OIS) is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the proliferative pool. Using combined genetic and bioinformatic analysis, we find that OIS is linked specifically to the activation of an inflammatory transcriptome. Induced genes included the pleiotropic cytokine interleukin-6 (IL-6), which upon secretion by senescent cells acted mitogenically in a paracrine fashion. Unexpectedly, IL-6 was also required for the execution of OIS, but in a cell-autonomous mode. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. Furthermore, we demonstrate that the transcription factor C/EBPbeta cooperates with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with arrested, p16(INK4A)-positive epithelium. We propose a model in which the context-dependent cytostatic and promitogenic functions of specific interleukins contribute to connect senescence with an inflammatory phenotype and cancer.

Comparison of the effects of 40% oxygen and two atmospheric absolute air pressure conditions on stress-induced premature senescence of normal human diploid fibroblasts

The pressure during hyperbaric oxygen treatment may increase oxygen toxicity via an augmented oxygen pressure in the gas. Nevertheless, only a few reports have been published on the effect of cells grown under 2 atmospheric absolute (ATA) pressure. To evaluate the effect of pressure on oxygen toxicity and to study effects in addition to oxygen toxicity, we designed an experiment to compare the effects of normobaric mild hyperoxia (NMH, 40% oxygen) and hyperbaric air condition (HA, air with 2 ATA) on human diploid fibroblasts (HDF) in a hyperbaric incubator. HDFs in both the NMH and the HA condition had a similar oxidative stress response and exhibited premature senescence. To investigate differences in gene profiling in cells grown in the NMH and HA conditions, samples from cells exposed to each condition were applied to microarrays. We found no expression difference in genes related to aging and deoxyribonucleic acid damage, but the expression of genes including cell adhesion, stress response, and transcription were significantly increased in fibroblasts that were responsive to pressure. Among 26 statistically reliable genes, the expression of apoptosis related genes such as ADAM22, Bax, BCL2L14, and UBD, as well as tumor suppressor-related genes like Axin2 and ATF, and also mitogen-activated protein kinase-related genes like mitogen-activated protein kinase kinase kinase 1, histamine receptor, and RAB24, were significantly changed in cells responsive to pressure-induced oxidative stress.

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

Transforming Growth Factor β1 Induces αvβ3 Integrin Expression in Human Lung Fibroblasts via a β3 Integrin-, c-Src-, and p38 MAPK-dependent Pathway

The Chemopreventive Agent Curcumin Is a Potent Radiosensitizer of Human Cervical Tumor Cells via Increased Reactive Oxygen Species Production and Overactivation of the Mitogen-Activated Protein Kinase Pathway

Cervical cancer is the second most common malignancy among women worldwide and is highly radioresistant, often resulting in local treatment failure. For locally advanced disease, radiation is combined with low-dose chemotherapy; however, this modality often leads to severe toxicity. Curcumin, a polyphenol extracted from rhizomes of the plant Curcuma longa, is a widely studied chemopreventive agent that was shown to have a low toxicity profile in three human clinical trials. Here, we show that pretreatment of two cervical carcinoma cell lines, HeLa and SiHa, with curcumin before ionizing radiation (IR) resulted in significant dose-dependent radiosensitization of these cells. It is noteworthy that curcumin failed to radiosensitize normal human diploid fibroblasts. Although in tumor cells, curcumin did not significantly affect IR-induced activation of AKT and nuclear factor-kappaB, we found that it caused a significant increase in the production of reactive oxygen species, which further led to sustained extracellular signal-regulated kinase (ERK) 1/2 activation. The antioxidant compound N-acetylcysteine blocked the curcumin-induced increased reactive oxygen species (ROS), sustained activation of ERK1/2, and decreased survival after IR in HeLa cells, implicating a ROS-dependent mechanism for curcumin radiosensitivity. Moreover, PD98059 (2'-amino-3'-methoxyflavone)-, PD184352- [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophynylthio)butadiene]-specific inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) blocked curcumin-mediated radiosensitization, demonstrating that the sustained ERK1/2 activation resulting from ROS generation leads to curcumin-mediated radiosensitization. Together, these results suggest a novel mechanism for curcumin-mediated radiosensitization involving increased ROS and ERK1/2 activation and suggest that curcumin application (either systemically or topically) may be an effective radiation modifying modality in the treatment of cervical cancer.

Interferon-inducible IFI16 protein in human cancers and autoimmune diseases

Interferon-inducible IFI16 protein (encoded by IFI16 gene located at 1q21 region) is a member of the p200-protein family. The family includes structurally and functionally-related mouse (for example, p202, p203, and p204 proteins) and human (for example, MNDA, AIM2, and IFIX) proteins. Increased expression of p200-family proteins in a variety of cells is known to inhibit cell cycle progression and modulate cell survival. Consistent with this role of p200-family proteins, increased expression of IFI16 protein in normal human diploid fibroblasts and prostate epithelial cells is associated with cellular senescence-associated permanent cell growth arrest. Furthermore, reduced or loss of IFI16 expression in cells is associated with the development of certain cancers, such as breast and prostate cancer. Interestingly, recent studies have provided evidence that the constitutive and interferon-induced expression of the IFI16 gene varies among individuals and may depend on the race. These studies raise the possibility that alterations (increases or decreases) in the expression of IFI16 protein may contribute to the development of human diseases. In this review, we discuss how our understanding of the regulation of IFI16 expression and its role in cell growth regulation will help elucidate the molecular mechanisms that contribute to the development of various human diseases.

Caspase‐8 dependent osteosarcoma cell apoptosis induced by proteasome inhibitor MG132

Many researchers have reported that proteasome inhibitors could induce apoptosis in a variety of cancer cells, such as breast cancer cell, lung cancer cell, and lymphoma cell. However, the effect of proteasome inhibitors on osteocsarcoma cells and the mechanisms are seldom studied. In this study, we found proteasome inhibitor MG132 was an effective inducer of apoptosis in human osteosarcoma MG-63 cells. On normal human diploid fibroblast cells, MG132 did not show any apoptosis-inducing effects. Apoptotic changes such as DNA fragment and apoptotic body were observed in MG132-treated cells and MG132 mostly caused MG-63 cell arrest at G(2)-M-phase by cell cycle analysis. Increased activation of caspase-8, accumulation of p27(Kip1), and an increased ratio of Bax:Bcl-2 were detected by RT-PCR and Western blot analysis. Activation of caspase-3 and caspase-9 were not observed. This suggests that the apoptosis induced by MG132 in MG63 cells is caspase-8 dependent, p27 and bcl-2 family related.

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/μm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to γ-rays or energetic carbon ions (108 keV/μm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than γ-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than γ-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.

Surveillance mechanism linking Bub1 loss to the p53 pathway

Bub1 is a kinase believed to function primarily in the mitotic spindle checkpoint. Mutation or aberrant Bub1 expression is associated with chromosomal instability, aneuploidy, and human cancer. We now find that targeting Bub1 by RNAi or simian virus 40 (SV40) large T antigen in normal human diploid fibroblasts results in premature senescence. Interestingly, cells undergoing replicative senescence were also low in Bub1 expression, although ectopic Bub1 expression in presenescent cells was insufficient to extend lifespan. Premature senescence caused by lower Bub1 levels depends on p53. Senescence induction was blocked by dominant negative p53 expression or depletion of p21(CIP1), a p53 target. Importantly, cells with lower Bub1 levels and inactivated p53 became highly aneuploid. Taken together, our data highlight a role for p53 in monitoring Bub1 function, which may be part of a more general spindle checkpoint surveillance mechanism. Our data support the hypothesis that Bub1 compromise triggers p53-dependent senescence, which limits the production of aneuploid and potentially cancerous cells.

Busulfan-induced senescence is dependent on ROS production upstream of the MAPK pathway

Induction of cellular senescence is a common response of a normal cell to a DNA-damaging agent, which may contribute to cancer chemotherapy- and ionizing radiation-induced normal tissue injury. The induction has been largely attributed to the activation of p53. However, the results from the present study suggest that busulfan (BU), an alkylating agent that causes DNA damage by cross-linking DNAs and DNA and proteins, induces senescence in normal human diploid WI38 fibroblasts through the extracellular signal-regulated kinase (Erk) and p38 mitogen-activated protein kinase (p38 MAPK) cascade independent of the p53-DNA damage pathway. The induction of WI38 cell senescence is initiated by a transient depletion of intracellular glutathione (GSH) and followed by a continuous increase in reactive oxygen species (ROS) production via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which leads to the activation of the Erk and p38 MAPK pathway. Incubation of WI38 cells with N-acetylcysteine (NAC) replenishes intracellular GSH, abrogates the increased production of ROS, ameliorates Erk and p38 MAPK activation, and attenuates senescence induction by BU. Thus, inhibition of senescence induction using a potent antioxidant or specific inhibitor of the Erk and p38 MAPK pathway has the potential to be developed as a mechanism-based strategy to ameliorate cancer therapy-induced normal tissue damage.

A Virus Causes Cancer by Inducing Massive Chromosomal Instability through Cell Fusion

Chromosomal instability (CIN) underlies malignant properties of many solid cancers and their ability to escape therapy, and it might itself cause cancer [1, 2]. CIN is sustained by deficiencies in proteins, such as the tumor suppressor p53 [3-5], that police genome integrity, but the primary cause of CIN in sporadic cancers remains uncertain [6, 7]. The primary suspects are mutations that deregulate telomere maintenance, or mitosis, yet such mutations have not been identified in the majority of sporadic cancers [6]. Alternatively, CIN could be caused by a transient event that destabilizes the genome without permanently affecting mechanisms of mitosis or proliferation [5, 8]. Here, we show that an otherwise harmless virus rapidly causes massive chromosomal instability by fusing cells whose cell cycle is deregulated by oncogenes. This synergy between fusion and oncogenes "randomizes" normal diploid human fibroblasts so extensively that each analyzed cell has a unique karyotype, and some produce aggressive, highly aneuploid, heterogeneous, and transplantable epithelial cancers in mice. Because many viruses are fusogenic, this study suggests that viruses, including those that have not been linked to carcinogenesis, can cause chromosomal instability and, consequently, cancer by fusing cells.

In vitro biological activity of a novel small-molecule inhibitor of polo-like kinase 1

Polo-like kinase 1 (PLK1) plays key roles in the regulation of mitotic progression, including mitotic entry, spindle formation, chromosome segregation, and cytokinesis. PLK1 expression and activity are strongly linked to proliferating cells. Many studies have shown that PLK1 expression is elevated in a variety of tumors, and high expression often correlates with poor prognosis. Using a variety of methods, including small-molecule inhibition of PLK1 function and/or activity, apoptosis in cancer cell lines, cell cycle arrest in normal cell lines, and antitumor activity in vivo have been observed. In the present study, we have examined the in vitro biological activity of a novel and selective thiophene benzimidazole ATP-competitive inhibitor of PLK1 and PLK3 (5-(5,6-dimethoxy-1H-benzimidazol-1-yl)-3-{[2-(trifluoromethyl)-benzyl]oxy}thiophene-2-carboxamide, called compound 1). Compound 1 has low nanomolar activity against the PLK1 and PLK3 enzymes and potently inhibits the proliferation of a wide variety of tumor cell lines. In the lung adenocarcinoma cell line NCI-H460, compound 1 induces a transient G(2)-M arrest, mitotic spindle defects, and a multinucleate phenotype resulting in apoptosis, whereas normal human diploid fibroblasts arrest in G(2)-M and show little apoptosis. We also describe a cellular mechanistic assay that was developed to identify potent intracellular inhibitors of PLK1. In addition to its potential as a therapeutic agent for treating cancer, compound 1 is also a useful tool molecule for further investigation of the biological functions of PLK1 and PLK3.

H2O2 Accelerates Cellular Senescence by Accumulation of Acetylated p53 via Decrease in the Function of SIRT1 by NAD+ Depletion

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD⁺-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H₂O₂ and examined DNA cleavage, depletion of intracellular NAD⁺, expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated β-galactosidase (SA-β-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H₂O₂, though no cell death was observed. NAD⁺ levels in TIG-3 cells treated with H₂O₂ were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD⁺ levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H₂O₂ treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H₂O₂ induced cell cycle arrest at 24h and increased SA-β-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.

Bystander responses in three-dimensional cultures containing radiolabelled and unlabelled human cells

Research on the radiation-induced bystander effect has been carried out mainly in 2-D tissue culture systems. This study uses a 3-D model, wherein apparently normal human diploid fibroblasts (AG1522) are grown in a carbon scaffold, to investigate the induction of a G(1) checkpoint in bystander cells present alongside radiolabelled cells. Cultures were simultaneously pulse-labelled with (3)H-deoxycytidine ((3)HdC) to selectively irradiate a minor fraction of cells, and bromodeoxyuridine (BrdU) to identify the radiolabelled cells. After thorough washing of cultures, iododeoxyuridine (IdU) was administered to detect proliferating bystander cells. The cultures were harvested at various times thereafter, and cells were reacted with two monoclonal antibodies specific to IdU/BrdU or BrdU, respectively, stained with propidium iodide, and subjected to multi-parameter flow cytometry. Cell-cycle progression was followed in radiolabelled cells (BrdU(+)) that were chronically irradiated by low energy beta particles emitted by DNA-incorporated (3)H, and in unlabelled bystander cells (BrdU(-)) by a flow cytometry based cumulative labelling index assay. As expected, radiolabelled cells were delayed, in a dose-dependent manner, in G(2) and subsequently G(1). No delay occurred in progression of bystander cells through G(1), when the labelled cells were irradiated at dose rates up to 0.32 Gy h(-1).

Critical Role of Recombinase (HsRAD51) in Genetic Instability in Multiple Myeloma.

A number of cancers, including multiple myeloma (MM), show a significant genetic instability that evolves with the progression of disease. The molecular basis for the generation of genetic diversity in cancer cells has therefore emerged as an important focus of investigation. We have previously shown that homologous recombination (HR), required for a variety of normal cellular processes, is dysregulated in multiple myeloma and leads to a progressive accumulation of genetic variation over time. Moreover the inhibition or stimulation of HR activity in MM cells, by altering components of the HR pathway, concordantly affects the acquisition of new genetic changes. Consistent with this, an elevated frequency of cancer is observed in patients whose cells have elevated rate of recombination. HsRAD51 recombinase, is a key molecule in HR pathway and its dysregulated expression can increase in HR activity, leading to increased rate of mutations and development or progression of cancer. Based on these data, we hypothesized that hsRAD51 promoter activity may be a target for carcinogenic agents and ability to measure the hsRAD51 promoter activity may provide a simpler method to assess oncogenic potential of test agents. To this end we constructed a mammalian expression vector containing firefly luciferase gene downstream of hsRAD51 promoter. This plasmid was transfected into normal human plasma cells, multiple myeloma cell lines (ARP, OPM1, U266), and normal human diploid fibroblasts. The cells were allowed to recover for 24 hrs and then treated with agents with carcinogenic or anti-oxidant activities, or control vehicles for 2 hrs; lysed and luciferase activity assessed. RAD51 promoter activity was elevated 7- to 1000-fold in MM cells relative to normal plasma cells. Exposure of MM cells to a carcinogen (nickel chloride) was able to further induce hsRAD51 promoter activity (2-fold) in MM cells with already over-expression of hsRAD51. However, the anti-oxidant tocopherol and cannabinol were able to reduce hsRAD51 promoter activity by 3- and 8-fold respectively, while vitamin C and Selenium had minimal effects. In normal diploid fibroblasts, although the exposure to a non-carcinogen, Sodium azide did not induce hsRAD51 promoter activity, the exposure to agents with known transforming ability, Mitomycin C and Nickel chloride, led to a significant induction of hsRAD51 promoter activity (2.5 and 3.5-fold respectively). Moreover, the ability of these chemicals to induce RAD51 promoter activity covaried with their ability to induce homologous recombination activity as measured by a novel plasmid based assay (r = 0.9). We also demonstrate that Induction of RAD51 and HR or its inhibition using siRNAs targeting hsRAD51 can inhibit acquisition of new mutations in MM cells as measured by change in genome-wide loss of heterozygosity (LOH) using the 100K single nucleotide polymorphism arrays. These preliminary results provides us with a simple and powerful method to measure oncogenic potential of known carcinogens and demonstrate a critical role for RAD51 in ongoing genomic instability in MM and a rationale to target RAD51 to prevent progression.

P53 Is Preferentially Recruited to the Promoters of Growth Arrest Genes p21 and GADD45 during Replicative Senescence of Normal Human Fibroblasts

Replicative senescence is the terminal growth arrest that most normal human cells enter into after a fixed number of divisions in vitro, limiting the proliferative potential of a cell and preventing genomic instability caused by critically short telomeres. Thus, senescence presents a tumor-suppressive mechanism and a barrier to tumor formation. However, senescent cells are inherently resistant to apoptosis and, as they accumulate in aging tissues, may contribute to organ dysfunction and promote tumor progression as part of the stromal environment. Replicative life span in normal human cells can be extended by inactivation of the tumor suppressor gene p53 or its direct target, the cyclin-dependent kinase inhibitor p21, suggesting a direct role for this pathway in senescence. However, p53 recruitment to promoters of target genes during replicative senescence has not been shown in live cells. In this study, we used chromatin immunoprecipitation to determine that p53 preferentially occupied the promoters of growth arrest genes p21 and GADD45 in senescent normal human diploid fibroblasts but not the promoters of other target genes that recruited p53 following doxorubicin-induced DNA damage, such as apoptosis regulators TNFRSF10b, TNFRSF6, and PUMA. This differential recruitment of p53 in senescent versus doxorubicin-treated fibroblasts was accompanied by differences in post-translational modification of p53. These data provide mechanisms for both the growth arrest mediated by p53 and the resistant nature of senescent cells to apoptosis despite p53 activity.

Busulfan Selectively Induces Cellular Senescence but Not Apoptosis in WI38 Fibroblasts via a p53-Independent but Extracellular Signal-Regulated Kinase-p38 Mitogen-Activated Protein Kinase-Dependent Mechanism

Busulfan (BU) is a unique alkylating agent that primarily targets slowly proliferating or nonproliferating cells in the body, leading to various normal tissue damage while killing leukemia cells. However, the mechanism(s) of action whereby BU injures normal cells has not been well defined and, therefore, was investigated in the present study by using the normal human diploid WI38 fibroblasts as a model system. We found that WI38 fibroblasts incubated with BU (from 7.5-120 microM) for 24 h underwent senescence but not apoptosis in a dose-independent manner, whereas cells incubated with 80 and 20 microM etoposide (Etop) were committed to apoptosis and senescence, respectively. The induction of WI38 cell senescence by Etop was associated with p53 activation and could be attenuated by down-regulation of p53 using alpha-pifithrin (alpha-PFT) or p53 small interference RNA (siRNA). In contrast, WI38 cell senescence induced by BU was associated with prolonged activation of extracellular signal-regulated kinase (Erk), p38 mitogen-activated protein kinase (p38), and c-Jun NH(2)-terminal kinase (JNK) and could be suppressed by the inhibition of Erk and/or p38 with PD98059 (2'-amino-3'-methoxyflavone) and/or SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole], respectively. However, inhibition of p53 with alpha-PFT or p53 siRNA or JNK with SP600125 (1,9-pyrazoloanthrone) failed to protect WI38 cells from BU-induced senescence. These findings suggest that BU is a distinctive chemotherapeutic agent that can selectively induce normal human fibroblast senescence through the Erk and p38 pathways.

The Ras-MAPK pathway downregulates Caveolin-1 in rodent fibroblast but not in human fibroblasts: implications in the resistance to oncogene-mediated transformation

Normal human diploid fibroblasts (HDFs) are refractory to oncogene-mediated transformations in vitro, compared with rodent fibroblasts. As successful oncogene-mediated transformations of normal HDFs have been reported using the human telomerase catalytic subunit, it has been considered that telomerase activity contributes to the species-specific transformability. However, these transformed HDFs are much less malignant compared with those of rodent cells, suggesting the existence of undefined mechanisms that render HDFs resistant to malignant transformation. Here, cDNA microarray analysis identified caveolin-1 as one of the possible cellular factors involved in such mechanisms. The mitogen-activated protein kinases (MAPK) pathway downregulates Caveolin-1 in rodent fibroblasts, transformed by coexpression of the SV40 early region and activated H-Ras. In contrast, the coexpression of these two oncogenes in HDFs failed to reduce the expression level of Caveolin-1. These results strongly suggest the presence of critical differences in events following the phosphorylation of ERK during the activation process of the MAPK signaling pathway between human and rodent cells, as the ERK protein was similarly phosphorylated in both systems. Furthermore, the small interfering RNA-mediated suppression of Caveolin-1 facilitated the oncogene-mediated transformation of normal HDFs, clearly indicating that the differences in the transformability between human and rodent cells are due, at least in part, to the mechanism responsible for the resistance to Ras-induced Caveolin-1 downregulation in HDFs.