International Journal of Oncology 46: 2299-2308, 2015

The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment

 

Laura Annovazzi1, Valentina Caldera1, Marta Mellai1, Chiara Riganti2, Luigi Battaglia3, Daniela Chirio3, Antonio Melcarne4 and Davide Schiffer1

1Neuro-Bio-Oncology Center, Policlinico di Monza Foundation (Vercelli), I-13100 Vercelli, Italy;

2Department of Oncology, University of Turin, I-10126 Turin, Italy;

3Department of Drug Science and Technology, University of Turin, I-10125 Turin, Italy;

4Department of Neurosurgery, CTO Hospital, Città della Salute e della Scienza, I-10126 Turin, Italy

 

Abstract

Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas.

PMID: 25892134

 

Supplement:

Glioblastoma multiforme (GBM) is the most lethal adult brain tumor. Despite aggressive treatment regimen, based on maximal safe surgery, radiation therapy, concomitant and adjuvant temozolomide (TMZ) chemotherapy, GBM always recurs and the prognosis remains poor with a median survival of 14-16 months (1).

The failure of therapies is due to the diffuse and infiltrative nature of the tumor, to the presence of the blood-brain barrier and to intrinsic or acquired resistance of glioma cancer cells. Since radio- and chemotherapies are genotoxic, many DNA repair mechanisms have been shown to contribute to development of such resistance.

A great evidence indicates that a subpopulation of cancer cells, the glioma stem cells (GSCs), characterized by stem cell-like properties, self-renewing, in vitro multilineage differentiation potential and in vivo tumorigenic capability, is responsible for therapeutic resistance and GBM relapse. It has been shown that GSCs possess an increased DNA repair capacity compared to the other tumor non-stem cells (2).

The progression of cells through the cell cycle is finely and strictly controlled. Defective cells, such as those carrying DNA mutations or damage, can be either repaired and survive or can be eliminated. Therefore, an enhanced as well as a decreased repair response to DNA damage can confer a survival advantage to cancer cells and allow tumor growth despite genotoxic radio- and chemotherapies.

In response to DNA double strand breaks (DSBs), that are the most serious genotoxic lesion, mammalian cells activate a transduction cascade of sensor and effector molecules, including ataxia telangiectasia mutated (ATM), H2AX histone, 53 binding protein 1 (53BP1) and Checkpoint 2 (Chk2), that leads finally to the arrest of cell cycle. Cells try to repair the damage by means of two main mechanisms, depending on the phase of the blocked cycle: homologous recombination (HR) and non-homologous end-joining (NHEJ). If repair fails because the damage is too extensive, apoptosis is triggered (Figure 1).

 

Figure 1

Figure 1. Scheme of cell checkpoint/repair signaling activation after DNA damage occurrence.

 

In this work we investigated the in vitro cytotoxicity of three commonly used chemotherapeutics, TMZ, doxorubicin (Dox) and paclitaxel (PTX), on GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying the DNA damage degree caused by drug action and the cell response, i.e. the arrest of cell cycle with the repair signaling activation or the entry into apoptosis.

NS and AC, obtained from surgically resected GBMs, were immunohistochemically and genetically characterized. NS cultures, obtained on a serum-free medium supplemented with growth factors, possess stemness properties and in vivo tumorigenicity capacity; they likely contain GSCs/progenitors. AC, cultured with serum, represent a more differentiated tumor cell status (3).

After exposure, the in vitro cytotoxicity of the three drugs was evaluated assessing the number of viable cells by Trypan blue dye exclusion assay.

TMZ, Dox and PTX were able to inhibit dose- and time-dependently cell proliferation and the cytotoxic effect resulted much more evident on AC than on NS.

As regards TMZ, we observed an almost complete resistance of NS to dosages of TMZ lower than 10 mM, that are concentrations similar to those reached by the drug in the cerebrospinal fluid after clinical administration. Our results suggested that susceptibility of cells to TMZ depends tightly on the status of O6-methylguanine-DNA methyltransferase (MGMT), the most potent mediator of chemoresistance to TMZ, and also on the status of TP53. Indeed, only two NS lines with hypermethylated MGMT gene promoter and wild-type TP53 displayed an IC50 (the drug concentration able to cause a 50% cell growth inhibition) value for TMZ < 50 mM. On the contrary, on most AC lines, TMZ concentrations < 50 mM were able to significantly reduce cell growth after 72 hours (h) exposure. We observed that, even after long treatments with high TMZ dosage (200 mM), a few cells survived and reacquired growth capacity within 1-2 months after treatment suspension. The effect of TMZ in NS was an arrest of proliferation rather than an increase of cell death, as revealed by the observations on apoptosis occurrence.

Dox inhibited proliferation of both NS and AC in a stronger manner in comparison with TMZ. After 72 h treatment with 5 mM Dox, 80% cells were dead.

Also PTX was able to inhibit cell proliferation both in NS and in AC, but, after 5-days treatment with 100 nM PTX, in NS a few single cells survived and reacquired growth capacity within one month after treatment suspension.

We evidenced by Comet assay the DNA damage occurrence in the GBM cells after treatments, proving that all the 3 tested drugs are genotoxic. It was observed that the length of DNA tails, i.e. the extent of damage, increased proportionally with drug doses and times (Figure 2).

 

Figure 2

Figure 2. Comet assay on a GBM NS cell line. In untreated cells no DNA tail is detectable. After treatment for 72 h with increasing doses of TMZ (50, 100, 200 mM), with 100 nM PTX or with 2 mM Dox, comet tails appear with a length proportional to the degree of the DNA damage.

 

The presence of DSBs was confirmed by immunofluorescence/immunocytochemistry analysis of g-H2AX histone, that is a marker of double strand lesions. A significant increase in g-H2AX foci was evident, both in NS and in AC, mostly after TMZ and Dox treatment. The number of positive foci resulted proportional to the drug doses and exposure times.

It was also possible to observe a moderate DNA damage after 72 h treatment with 100 nM PTX, both in NS and in AC.

The apoptotic process elicited by drug exposure was evaluated by TUNEL assay. It was a late phenomenon, observable only after almost 72 h from the beginning of treatments. Cell death was more evident in AC than in NS, but in both cases the frequency of apoptosis after treatments with the 3 drugs does not explain the level of reduction of viable cells. Mainly as for TMZ, a reduction of cell proliferation and of clonogenic growth but not a correspondent increase of apoptosis takes place.

We then evaluated the activation of the factors of DNA damage/repair cascade by immunofluorescence on GBM cell lines and by immunohistochemistry on GBM and low-grade glioma tissues. Analysis was performed on NS and AC following TMZ and PTX treatments at various doses and times. We could note in some untreated NS lines a basal expression of the checkpoint/repair proteins, mostly of p-ATM, p-Chk2, DNA-PKcs, which was further enhanced following DNA insults. After NS treatment with 100 mM TMZ for 48 h, the expression of all the sensor, transducer and effector proteins of damage/repair, with the exception of RAD51, was evident (Figure 3). After DNA injury, however, also AC were able to trigger a repair response, although at a minor extent. Sensors of damage, i.e. g-H2AX, p-ATM, p-53BP1, were the first proteins to activate themselves and they decreased with time, as the effectors, mainly those of NHEJ system, increased and repair of damage took place.

 

Figure 3

Figure 3. Study of checkpoint/repair cascade by immunofluorescence on a GBM NS cell line, before and after treatment with 100 mM TMZ for 48 h.

 

 

It can be hypothesized that reduced proliferation and maintenance of a quiescent status joined with a higher repair capacity allow malignant glioma cells to become insensitive to chemotherapy.

Immunohistochemical investigation on tumor tissues revealed that the key proteins of the damage signaling and of NHEJ repair system are constitutively expressed in the GBM specimens, particularly in proliferation areas and in perinecrotic pseudopalisades. All the markers were almost negative in the low-grade glioma tissues analyzed, with the exception of p-ATM and g-H2AX, which were poorly positive at level of mitoses. DNA damage response signaling is an important factor in both tumorigenesis and treatment response of gliomas. It is an early event at the beginning of gliomagenesis, initially limiting cell proliferation and functioning as a protective mechanism against oncogenic progression (4). The aberrant constitutive activation of repair pathways found in GBM tumors can be considered as an early response to genomic instability, that, subsequently, becomes an advantage for cancer cells driving their resistance to exogenous insults caused by genotoxic therapies.

Importance of the study: This study showed the ability of GBM NS, which have been proved to contain GSCs/progenitors, to respond to three chemotherapeutics, blocking cell cycle and activating DNA repair mechanisms. Our results evidenced that NS are significantly more chemoresistant than the more differentiated counterpart of AC. We demonstrated that after TMZ exposure in NS cultures the depletion of cells was never complete even with high drug doses and that cells resisted to drug action stopping proliferation and surviving in a status of metabolic inertia. Furthermore, NS seemed to possess an enhanced capacity to sense and repair DNA damage compared with AC: the key-molecules p-ATM, p-Chk2, g-H2AX, DNA-PK appeared up-regulated in NS, whereas AC lines resulted more sensitive to drug treatments, presenting a greater reduction of viable cell number and a lower expression of repair cascade markers.

Moreover, we demonstrated that Dox and PTX have a strong anti-proliferative effect on glioma cells, indicating that these drugs, once the way to reach the tumor site is found, would be effective cytotoxic agents, potentially useful in the treatment of GBM. In this regard, our group is studying innovative nanoparticle formulations to vehicle these non-lipophilic drugs across the blood-brain barrier towards the tumor site (5).

Proteins involved in the sensing and repair of DNA damage after exposure to genotoxic agents can be attractive targets and exogenous modulation of DNA checkpoint kinases and repair effectors can be a useful means to overcome resistance and increase sensitivity of tumor cells to treatments. Controlled targeted inhibition of the DNA damage/repair factors, such as ATM, Chk2 and DNA-PK, combined with standard radio- and chemotherapy would represent a potentially efficacious strategy that may improve overall survival of patients.

 

References

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Contact:

Laura Annovazzi, PhD

Senior Researcher

Research Center, Policlinico di Monza Foundation

Via Pietro Micca 29, 13100 Vercelli, Italy

laura.annovazzi@cnbo.it

 

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