Content uploaded by Chuck Gomer
Author content
All content in this area was uploaded by Chuck Gomer
Content may be subject to copyright.
A preview of the PDF is not available
Photodynamic Therapy Mediated Induction of Early Response Genes
Abstract and Figures
Photodynamic therapy (PDT) generates reactive oxygen species which initiate the cytotoxic events of this tumor treatment. We demonstrate that PDT mediated oxidative stress induced a transient increase in the early response genes c-fos, c-jun, c-myc, and egr-1 in murine radiation-induced fibrosarcoma cells. Incubation of exponentially growing cells with porphyrin based photosensitizers in the dark also induced an increase in mRNA levels of early response genes. However, the xanthine photosensitizer, rose bengal, produced increased c-fos mRNA levels only following light treatment. Nuclear runoff experiments confirmed that the induction of c-fos mRNA is controlled in part at the level of transcription. Likewise, a chloramphenicol acetyltransferase reporter construct containing the major c-fos transcriptional response elements was inducible by porphyrin and PDT. Signal transduction pathways associated with PDT mediated c-fos activation were examined by treating cells with protein kinase inhibitors. Staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine inhibited PDT mediated c-fos activation while N-(2-guanidinoethyl)-5-isoquinoline-sulfonamide had no effect. In addition, quinacrine, which can inhibit phospholipase activity, blocked PDT induced c-fos mRNA expression. These results suggest that photosensitizer mediated oxidative stress acts through protein kinase-mediated signal transduction pathway(s) to activate early response genes.
Figures - uploaded by Chuck Gomer
Author content
All figure content in this area was uploaded by Chuck Gomer
Content may be subject to copyright.
... Previous studies indicated that PDT might induce cancer cell survival pathway activation. For example, within perihilar cholangiocarcinoma (QBC939) cells, sublethal PDT (LC50) led to the alteration of survival signaling pathways such as HIF-1, NF-κB, AP-1, and heat shock factor (HSF) (Luna et al., 1994;Broekgaarden et al., 2015;Weijer et al., 2015Weijer et al., , 2016. PDT-treated QBC939 cell line also exhibited decreased protein levels related to the EGFR pathway, especially at LC90 (Weijer et al., 2017). ...
... Indeed, PDT treatment could alter a range of survival pathways, including HIF-1, NF-κB, AP-1, HSF, and EGFR (Luna et al., 1994;Broekgaarden et al., 2015;Weijer et al., 2015Weijer et al., , 2016Weijer et al., , 2017. In the present study, by cross-checking online microarray expression profiles reporting PDT-altered genes, we found that KLF10, a transcription factor, could be significantly induced by PDT treatment, which was further evidenced by the observation that PDT treatment upregulated KLF10 mRNA expression and protein levels in gemcitabineresistant cholangiocarcinoma cells. ...
Cholangiocarcinoma is a relatively rare neoplasm with increasing incidence. Although chemotherapeutic agent such as gemcitabine has long been used as standard treatment for cholangiocarcinoma, the interindividual variability in target and drug sensitivity and specificity may lead to therapeutic resistance. In the present study, we found that photodynamic therapy (PDT) treatment inhibited gemcitabine-resistant cholangiocarcinoma cells via repressing cell viability, enhancing cell apoptosis, and eliciting G1 cell cycle arrest through modulating Cyclin D1 and caspase 3 cleavage. In vivo , PDT treatment significantly inhibited the growth of gemcitabine-resistant cholangiocarcinoma cell-derived tumors. Online data mining and experimental analyses indicate that KLF10 expression was induced, whereas EGFR expression was downregulated by PDT treatment; KLF10 targeted the EGFR promoter region to inhibit EGFR transcription. Under PDT treatment, EGFR overexpression and KLF10 silencing attenuated the anti-cancer effects of PDT on gemcitabine-resistant cholangiocarcinoma cells by promoting cell viability, inhibiting apoptosis, and increasing S phase cell proportion. Importantly, under PDT treatment, the effects of KLF10 silencing were significantly reversed by EGFR silencing. In conclusion, PDT treatment induces KLF10 expression and downregulates EGFR expression. KLF10 binds to EGFR promoter region to inhibit EGFR transcription. The KLF10/EGFR axis participates in the process of the inhibition of PDT on gemcitabine-resistant cholangiocarcinoma cells.
... A variety of molecular signaling pathways have been identified in the adaptive responses of cancer cells that enable cells to cope with the effects of PDT [13]. These are mostly intrinsic pathways that implicate the transcription factors nuclear factor (erythroid-derived 2)-like 2 (NRF2), hypoxia-inducible factor 1 (HIF-1), nuclear factor-κB, Activator protein 1 (AP-1), and heat shock factors (HSFs) in mediating reduced sensitivity to PDT [13,[29][30][31][32][33][34][35][36]. The results of this study clearly identified MET signaling as an additional auto-and paracrine survival pathway in cancer cells that can be induced and sustained by HGF secretion from perishing stromal cells. ...
Extensive desmoplasia is a hallmark of pancreatic ductal adenocarcinoma (PDAC), which frequently associates with treatment resistance. Recent findings indicate that a combination of photodynamic therapy and the multi-kinase inhibitor cabozantinib achieved local tumor control and a significant decrease in tumor metastases in preclinical PDAC models, but the underlying therapeutic mechanisms remain unclear. This study elucidates the molecular basis of this multi-agent regimen, focusing on the role of MET signaling. Since MET activation stems from its interaction with hepatocyte growth factor (HGF), which is typically secreted by fibroblasts, we developed heterotypic PDAC microtumor models that recapitulate these interactions. In these models, MET signaling can be constitutively activated through paracrine and autocrine mechanisms. Photodynamic therapy caused significant elevations in HGF secretion by fibroblasts, suggesting it plays a complex role in the modulation of the paracrine HGF–MET signaling cascade in desmoplastic tumors. Blocking MET phosphorylation with adjuvant cabozantinib caused a significant improvement in photodynamic therapy efficacy, most notably by elevating spheroid necrosis at low radiant exposures. These findings highlight that adjuvant photodynamic therapy can augment chemotherapy efficacies, and potentially achieve improved management of desmoplastic PDAC in a more tolerable manner.
... Apart from the apoptosis/necrosis induced by PDT, many other molecular changes have been documented in cells undergoing PDT. These range from activation of transcription factors [58,59], stimulation of protein kinases [60], expression of stress proteins [61,62], production of cytokines [63][64][65] and prostaglandins [52,66], and oxidation of lipids [67]. ...
Ovarian cancer (OvCa) is the leading cause of gynecological cancer-related deaths in the United States, with 5-year survival rates of 15-20% for stage III cancers and 5% for stage IV cancers. Standard of care for advanced OvCa involves surgical debulking of disseminated disease in the peritoneum followed by chemotherapy. Despite advances in treatment efficacy, the prognosis for advanced stage OvCa patients remains poor and the emergence of chemoresistant disease localized to the peritoneum is the primary cause of death. Therefore, a complementary modality that is agnostic to typical chemo- and radio-resistance mechanisms is urgently needed. Photodynamic Therapy (PDT), a photochemistry-based process, is an ideal complement to standard treatments for residual disease. The confinement of the disease in the peritoneal cavity makes it amenable for regionally localized treatment with PDT. PDT involves photochemical generation of cytotoxic Reactive Molecular Species (RMS) by non-toxic photosensitizers (PS) following exposure to non-harmful red light, leading to localized cell death. However, due to the complex topology of sensitive organs in the peritoneum, diffuse intra-abdominal PDT induces dose-limiting toxicities due to non-selective accumulation of PS in both healthy and diseased tissue. In an effort to achieve selective damage to tumorous nodules, targeted PS formulations have shown promise to make PDT a feasible treatment modality in this setting. This targeted strategy involves chemical conjugation of PS to antibodies, referred to as PhotoImmunoConjugates (PICs), to target OvCa specific molecular markers leading to enhanced therapeutic outcomes while reducing off-target toxicity. In light of promising results of pilot clinical studies and recent preclinical advances, this review provides the rationale and methodologies for PIC-based PDT, or Photo-immunotherapy (PIT), in context of OvCa management.
... The effects of different PDT approaches on c-Myc status have been described previously for other cancer cell types, often with contrasting results. In some cases, expressed c-Myc mRNA or protein steadily increased after PDT (60,61), whereas in other cases, it decreased (62-64), but no rational explanations were offered. In most of these studies, c-Myc was not monitored for more than 4 -6 h after irradiation, whereas we ...
Endogenous nitric oxide (NO) generated by inducible NO synthase (iNOS) promotes glioblastoma cell proliferation and invasion, and also plays a key role in glioblastoma resistance to chemotherapy and radiotherapy. Non-ionizing photodynamic therapy (PDT) has anti-tumor advantages over conventional glioblastoma therapies. Our previous studies revealed that glioblastoma U87 cells upregulate iNOS after a photodynamic challenge and that resulting NO not only increased resistance to apoptosis, but rendered surviving cells more proliferative and invasive. These findings were largely based on the effects of inhibiting iNOS activity and scavenging NO. Demonstrating now that iNOS expression in photostressed U87 cells is mediated by NF-κB, we hypothesized that (i) recognition of acetylated lysine (acK) on NF-κB p65/Rel A by bromodomain and extra-terminal (BET) protein Brd4 is crucial, and (ii) by suppressing iNOS expression, a BET inhibitor (JQ1) would attenuate the negative effects of photostress. The following evidence was obtained: (i) Like iNOS, Brd4 protein and p65-acK levels increased several fold in photostressed cells; (ii) JQ1 at minimally toxic concentrations had no effect on Brd4 or p65-acK upregulation after PDT, but strongly suppressed iNOS, survivin, and Bcl-xL upregulation, along with the growth and invasion spurt of PDT-surviving cells; (iii) JQ1 inhibition of NO production in photostressed cells closely paralleled that of growth/invasion inhibition; (iv) At 1% the concentration of iNOS inhibitor 1400W, JQ1 reduced post-PDT cell aggressiveness to a far greater extent. This is the first evidence for BET inhibitor targeting of iNOS expression in cancer cells and how such targeting can markedly improve therapeutic efficacy.
... The mechanism of the PDT-mediated cytotoxic effect is still not fully understood. It was reported that PDT-induced oxidative stress could increase the expression of early response genes, the cellular stress protein and heme oxygenase (Luna et al. 1994). Moreover, it is widely acknowledged that cell cycle arrest is an important cause of cell apoptosis. ...
Chlorophyll has always been used as the leading compound for photodynamic therapy drug development. In this paper, a novel methyl pyropheophorbide-a- derived photosensitizer, 3-acetyl-3-devinyl-13¹-dicyanomethylene-pyropheophorbide-a was synthesized through modifications at C-13¹, C-3, and C-17 positions of methyl pyropheophorbide-a. The compound exhibited a longer wavelength absorption at 713 nm (in methanol) than that of methyl pyropheophorbide-a (667 nm) due to the enlarged the aromatic conjugation system by dicyanomethylene, allowing it to be potential in deep tumor treatment. Moreover, benefiting from the carboxylic group at C-17 and the acetyl group at C-3, the title compound was endowed with better water solubility than that of methyl pyropheophorbide-a. Detailed in vitro photodynamic therapy research showed ADCPPa could be uptaken by cancer cells successfully and killed the cancer cells more efficiently than the leading compound methyl pyropheophorbide-a under light (light dose 10 J/cm²) due to the high singlet oxygen quantum yield (65.98%). The excellent anti-photobleaching ability (degradation rate 1.6% in 10 min) also boosted its potential in practical application. In addition, the research has disclosed that during photochemical processes of photodynamic therapy, the formation of singlet oxygen after photodynamic therapy treatment played a major role, comparing with the formation of superoxide anion and radicals. Finally, the real time quantitative polymerase chain reaction (RT-qPCR) experiments have showed that the target compound has important regulating effect on expression of CDK2 and Survivin, consequently leading to apoptosis and cell death.
... PDT is accompanied by oxidative stress (19). Oxidative stress is a biological phenomenon whereby reactive oxygen species are produced and subsequently cause cellular damage. ...
Protoporphyrin IX (PpIX) levels are crucial to the antitumor action of photodynamic therapy (PDT). In the present study, the underling molecular mechanisms for the variation in PpIX levels in ovarian cancer cells were investigated. Five ovarian cancer cell lines were subcutaneously grafted onto the backs of nude mice. Once tumors had developed, 5-aminolevulinic acid methyl ester hydrochloride (methyl-ALA) was administered intraperitoneally and the tumor was irradiated twice/week. PpIX levels in the tumor were assayed using high-performance liquid chromatography. Enzymes involved in heme synthesis and degradation were screened using a microarray technique. Expression of the glutathione transferase Omega-1 (GSTO1) gene involved in the conversion of PpIX into heme in cells was quantified using the reverse transcription-quantitative polymerase chain reaction. In HTOA, HRA and DISS cells, PDT resulted in significant tumor shrinkage in comparison with the controls. In MCAS and TOV21G cells, no significant alterations in tumor growth were identified compared with the untreated cells. PpIX levels increased significantly in HTOA, DISS and HRA cells compared with in MCAS and TOV21G cells. A comparison of genetic profiles using PDT-sensitive DISS cells and PDT-resistant MCAS cells indicated that MCAS cells exhibited significantly increased levels of δ-aminolevulinate synthase (a rate-limiting enzyme in heme synthesis), heme oxygenase 2 (an enzyme that degrades heme into biliverdin), and biliverdin reductase B (an enzyme that reduces biliverdin into bilirubin) in comparison with DISS cells. The level of GSTO1 expression in HTOA, HRA and DISS cells was ~2.5-fold that in MCAS and TOV21G cells. Sensitivity to PDT is related to PpIX levels in cells. The results of the present study suggested that PpIX tends not to accumulate in PDT-resistant cells despite active heme synthesis and degradation, and that high levels of GSTO1 expression are associated with increased sensitivity to PDT.
... PDT-mediated oxidative stress induces a transient increase in the downstream early response genes c-fos, c-jun, c-myc, and egr-1 (59). Assays of kinase activity have provided clues regarding the upstream molecules expressed and/or activated in cells following PDT (46,60). ...
Porphyromonas gingivalis has been implicated in the development of periodontal diseases and the current treatments used are not entirely successful in eradicating periodontopathogens from the pocket. Photodynamic therapy has been successfully used in the treatment of a number of cancers and has been used in vitro to kill a variety of bacteria. Lethal photosensitisation of Por. gingivalis using toluidine blue O (TBO) with Helium/Neon (HeNe) laser light was compared to lethal photosensitisation using aluminium disulphonated phthalocyanine (AIPCS2) with gallium aluminium arsenide (GaAs) laser light. It was found that there were substantial reductions in viable counts using TBO/HeNe laser light and when using AIPcS2/GaAs laser light. Substantial kills were obtained when lethal photosensitisation was carried out under conditions most likely to be encountered in the periodontal pocket (presence of serum, increasing pH values, different Por. gingivalis strains and biofilm-grown bacteria). The involvement of singlet oxygen and hydroxyl radicals was investigated by carrying out lethal photosensitisation in the presence of the singlet oxygen enhancer, deuterium oxide. It was found that singlet oxygen and possibly hydroxyl radicals are involved in the killing of Por. gingivalis. Distribution studies using 3H-TBO showed that most of the 3H-TBO was bound to the outer membrane and SDS-PAGE analysis showed that there were alterations to the outer and plasma membrane proteins from cells sensitised with TBO and exposed to laser light. It was also found that there was DNA degradation as a result of lethal photosensitisation. The final part of the study involved conjugating antibody against surface components of Por. gingivalis with TBO and specifically to target Por gingivalis to light-induced killing when in the presence of commensal bacteria or host tissue. When sensitised with the antibody/TBO conjugate and exposed to laser light, there were no significant reductions in viable counts of Streptococcus sanguis or human gingival fibroblasts, whereas all of the Por. gingivalis present were killed. In conclusion, Por. gingivalis can be killed effectively (100%) when sensitised with 82 μM TBO at an energy dose of 4.4 J. Type II and possibly type I mechanisms are involved in the killing of Por gingivalis and the outer and plasma membrane proteins and DNA are adversely affected by lethal photosensitisation. Damage to oral commensal organisms and oral host tissue can be avoided by using laser light in combination with TBO conjugated to antibody against Por gingivalis surface components.
Nanocarriers sensitive to near infrared light (NIR) are useful templates for chemo-photothermal therapy (PTT)
and imaging of tumors due to the ability to change the absorbed NIR energy to heat. The conventional photo-absorbing
reagents lack the efficient loading and release of drug before reaching the target site leading to insufficient
therapeutic outcomes. To overcome these limitations, the surface of nanocarriers can be modified with different
polymers with wide functionalities to provide systems with diagnostic, therapeutic, and theranostic capabilities.
Among various polymers, polydopamine (PDA) has been more interested due to complex structure with
various chemical moieties, and the capacity to be used through different coating mechanism. In this review, we
describe the complex structure, chemical properties, and coating mechanisms of PDA. Moreover, the advantage
and surface modification of some relevant nanosystems based on carbon materials, gold, iron oxide, manganese,
and upconverting nanomaterials by using PDA will be discussed, in detail.
Photodynamic therapy (PDT) uses light-activated drugs (photosensitizers) for the treatment of neoplastic and non-neoplastic diseases. Administration of the photosensitizer constitutes the first step in the PDT process. Then, following a waiting period (minutes to days) to allow for selective accumulation in the target tissue, the sensitizer is activated via light (usually from a laser) of a wavelength matching a prominent absorption resonance in the red or near-infrared part of the visible spectrum. Absorption of this light by the photosensitizer results in photochemical processes which ultimately produce the cytotoxic species (e.g., singlet molecular oxygen) responsible for the biological damage.
Singlet oxygen, a metastable state of normal triplet oxygen, has been identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet oxygen trap is not toxic to the cells nor does it absorb the light responsible for hematoporphyrin activation. We have found that the singlet oxygen-trapping product, o-dibenzoylbenzene, is formed nearly quantitatively intracellularly when both the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of the TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet oxygen-trapping product indicates that singlet oxygen is the probable agent responsible for toxicity in this system.
Singlet oxygen, a metastable state of normal triplet oxygen, has been identified as the cytotoxic agent that is probably responsible for in vitro inactivation of TA-3 mouse mammary carcinoma cells following incorporation of hematoporphyrin and exposure to red light. This photodynamic inactivation can be completely inhibited by intracellular 1,3-diphenylisobenzofuran. This very efficient singlet oxygen trap is not toxic to the cells nor does it absorb the light responsible for hematoporphyrin activation. We have found that the singlet oxygen-trapping product, o-dibenzoylbenzene, is formed nearly quantitatively intracellularly when both the furan and hematoporphyrin are present during illumination but not when only the furan is present during illumination. The protective effect against photodynamic inactivation of the TA-3 cells afforded by 1,3-diphenylisobenzofuran coupled with the nearly quantitative formation of the singlet oxygen-trapping product indicates that singlet oxygen is the probable agent responsible for toxicity in this system. © 1976, American Association for Cancer Research. All rights reserved.
We examined the effect of quinacrine, an anti-malarial drug which inhibits phospholipase A2, on phorbol myristate acetate (PMA) stimulated alveolar macrophage oxygen radical secretion. This drug suppressed 30% of radicals release, which was 70% of the amount inhibited by superoxide dismutase, a specific inhibitor of oxygen radicals. In addition, this reduction was at the same magnitude as dexamethasone. This and previous results on other inflammatory cells support the assumption that quinacrine may have a beneficial effect on bronchial asthma.
GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal level of GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,00 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down-regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations.
Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.
We have compared the mechanisms of the transcriptional induction of c-fos in mouse epidermal cells JB6 (clone 30) by an extracellular burst of active oxygen of the type produced by inflammatory phagocytes to induction by serum and phorbol ester. All three inducers elicit a characteristic immediate early response of c-fos which is inhibited by the protein kinase inhibitor H7 but enhanced by the protein synthesis inhibitor cycloheximide. Experiments with stable transfectants containing fos 5' upstream regulatory sequences linked to an HSV-tk-chloram-phenicol-acetyl-transferase reporter construct indicate that the joint dyad symmetry element-AP-1 motifs exert the most potent enhancer effect in response to active oxygen as well as serum. It is concluded that the different signal transduction pathways used by these inducers converge to the same 5' regulatory sequences of c-fos. In contrast to these common features only active oxygen induction of c-fos required the poly-ADP-ribosylation of chromosomal proteins. The inhibitors of ADP-ribose transferase benzamide and 3-amino-benzamide suppressed the elongation of the c-fos message and the de novo synthesis of nuclear factors, among them c-Fos and c-Jun, which bind to the fos-AP-1 motif in vitro only following stimulation with active oxygen. No active oxygen-induced change was observed in the protein complex which binds to an oligonucleotide containing the SIF and dyad symmetry element motifs in vitro. The presence of Fos and Jun proteins was detected in this complex. Only active oxygen, but not serum or phorbol ester, induces DNA breakage. We propose that poly-ADP-ribosylation is required because it participates in the repair of DNA breaks which interfere with transcription. We observed that Fos protein is weakly poly-ADP-ribosylated in response to active oxygen, but the functional role of this modification remains unclear.
The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. However, the signaling events responsible for these effects remain unclear. The present studies have examined the effects of M-CSF on potential signaling pathways involving expression of the jun and fos early response genes. Low levels of c-jun transcripts were detectable in resting human peripheral blood monocytes. Treatment of these cells with 10(3) units/ml human recombinant M-CSF was associated with rapid and transient increases in c-jun mRNA levels. Nuclear run-on assays and mRNA stability studies demonstrated that M-CSF regulates c-jun expression by both an increase in transcription rate and a prolongation in the half-life of c-jun transcripts. M-CSF treatment was also associated with a rapid induction of the jun-B gene, although expression of this gene was prolonged compared to that of c-jun. We further demonstrate that M-CSF increases c-fos mRNA levels in human monocytes through control at both the transcriptional and posttranscriptional levels. Maximal induction of the c-fos gene was followed by that for the fos-B gene. Moreover, M-CSF-induced expression of the fos-related gene, fra-1, was delayed compared to that for both c-fos and fos-B. Taken together, the results indicate that M-CSF treatment is associated with differential activation of multiple members of the jun/fos family and that expression of these genes could contribute to nuclear signaling mechanisms that regulate a specific program of monocyte differentiation.
Oxidative stress plays an important role in various types of cell injury and tumor promotion. Cells respond to oxidative stress in many ways including changes in membrane organization, ion movements, and altered gene expression, all of which contribute to the subsequent fate of affected cells. In this study, we investigated the expression of the proto-oncogenes c-fos, c-myc, and c-jun, which play a key role in proliferation and differentiation, using primary cultures of rat proximal tubular epithelium exposed to oxidative stress generated by the xanthine/xanthine oxidase system. This system generates superoxide and H2O2 in the extracellular space stimulating the release of active oxygen species from inflammatory cells. c-fos mRNA was expressed within 15 min, peaked at 30 min, and returned to constitutive levels by 3 h. c-jun mRNA began to rise after 30 min, peaked at 120 min, and remained above the constitutive levels up to 180 min. c-myc mRNA expression was less affected by the treatment, with levels increasing gradually over the 180 min period. The expression of c-fos was inhibited by superoxide dismutase but not by catalase and was super-induced by cycloheximide. H2O2 alone did not induce any c-fos mRNA in this system. Chelation of extracellular ionized calcium by EGTA or of intracellular ionized calcium by Quin 2/AM resulted in a marked decrease of c-fos expression. Two protein kinase C inhibitors, H-7 and staurosporine, partly diminished the expression of c-fos, whereas a third, 2-aminopurine, which has a broader spectrum of inhibiting protein kinases, almost completely abolished it. A poly ADP-ribosylation inhibitor, 3-aminobenzamide, had no effect on c-fos expression in this system. Our results show that oxidative stress provokes sequential expression of c-fos, c-jun, and c-myc, mRNA in this order. This c-fos expression appears to be largely controlled by calcium ion movement, which could include protein kinase C activation. Another protein kinase or kinases also appear to play an important role.