Kamran Alimoghaddam MD

Chloroquine is an amine that is widely used for increasing transfection of genes into cells.¹ It is a weak base with different effects on cellular physiology² and entry of enveloped RNA viruses into cells.

As a weak base, it rapidly enters cells within seconds, accumulating inside the acidic parts of the cell (including lysosomes and endosomes).

Because of this accumulation inside the cells, some vacuoles appear. Accumulation of this weak base inside the cells leads to inhibition of lysosomal enzymes that require an acidic pH (~ 4 – 5) and also prevents fusion of endosomes and lysosomes, thereby preventing the degradation of ingested proteins and related foreign materials.^{2 – 4}

Chloroquine also exerts toxic effects on cells that are linearly related to the concentration of the drug and the duration of exposure. It also prevents cellular protein synthesis and degrades certain cellular enzymes.^{3 – 5}

Entry of enveloped RNA viruses (including retroviruses) into cells can be pH-dependent or pH- independent. For pH-dependent viral entry, the virus attaches itself to the receptor on the surface of the cell and is engulfed inside an endosome. Following reduction of the endosome, conforma-tional changes occur in the viral receptor. This is followed by fusion of the envelope and endosomal surface, aiding the entry of the virion particle into the cytosol. Amines and other substances that prevent acidification of the endosome prevent both the exit of the original virus and entry of a new viral particle.^{6 – 9}

For pH-independent viral entry, induction of conformational changes in the virus receptor are not required. The virus enters the cell directly from the cell surface, without any need for endosomal engulfment, or enters the cell so rapidly after engulfment that any associated pH changes are unnecessary. Amines (ammonium chloride, aman-tadine and chloroquine) cannot prevent entry of these viruses into cells.^10,11

Most of the mammalian retroviruses use a pH-independent mechanism,¹⁰ but some, such as ecotropic murine Moloney retrovirus, enter most cells as pH-dependent particles. However, in certain cell lines, it enters via a pH-independent mechanism.¹⁰ The mechanism of entry is depend-ent on both the virus and the cell type.

In most studies, chloroquine has been used as an inhibitor of virus entry—a tool to determine the mechanism of virus entry. In some studies, it was successfully used to augment the transduction of retrovirus-based vectors. Hence, many researchers concluded that chloroquine increased transduction efficiency by prevention of lysosomal digestion of retrovirus inside cells, thus rescuing some of the viral particles, thereby increasing transduction efficiency.¹²

Chloroquine has other effects on cells, which include inhibition of synthesis of proteases, phospholipases and steroids, basal DNA synthesis and alteration of membrane fluidity. Also, it binds to the cell surface and membrane phospholipids,⁴ and is toxic to several cell types.

In this study, the effect of chloroquine on transduction and associated toxicity of baboon CD34 cells and a variety of cell lines was studied.

We used PG13 packaging cell line containing a GALV pseudotyped (gibbon ape leukemia virus) MNDEGFPSN vector (H.P. Kiem Lab, Fred Hutchinson Cancer Research Center, Seattle, WA, USA) to study expression of the GFP (green fluorescence protein), 48 hours after transduction. For production of vector after transfer of 5.5 x 10⁶ to 15-cm dishes, on day 3 and after subconfluency of the cell cultures, the medium was changed with a minimum of applicable medium (15 mL), and the vector was harvested, 12 hours later, for a total of three times. After filtering, the vector preparations were transferred to a –70° freezer and used within 2 months after harvesting. The multiplicity of infection (MOI) from these harvests was about 3.

Transduction

Cell line transductions were performed in the presence of 8 µg/mL of protamine sulfate with different concentrations of chloroquine (50 µmol – 100 µmol). Time of exposure to the drug ranged from about 6 – 12 hours. After transduction, the medium was changed after washing the wells with phosphate-buffered saline (PBS), and the cells were refed with DMEM or RPMI. Forty-eight hours after transduction, the expression of GFP was read by a flow activated cell sorter (FACS) machine. In some of the samples, chloroquine was added 3 hours after beginning transduction for discrimination between toxicity or entry block, and some Hela cells were also transduced in the presence or absence of chloroquine at pH 5.8, to study the effects of pH changes on transduction. We prepared DMEM pH 5.0 by the addition of HCL, and then added 1 mL of the acidic medium to a subset of the wells that were untreated or pretreated with chloroquine for 15 minutes. After addition of an equal volume of vector containing normal pH DMEM, the final pH reached 5.8. After exposure of the cells to these conditions, the medium was changed by adding fresh normal pH DMEM. We repeated transduction studies on cell lines three times for each cell line, and each study was repeated at least once. All of the values obtained are shown as the mean of the transduction efficiencies (the duplicated results of the range in each experience were very close). However, we are not reporting results of the entire range for simplicity of reporting.

the drug for 6 – 24 hours. After 48 hours and after trypsinization of the cells and tryptan blue exclusion, the cells were counted and compared with the number of cells in the untreated wells. Further, on FACS analysis, we stained the cells with propidum iodide to exclude severely injured cells from the study.

of chloroquine. Transduction efficiencies differed in different cell lines in response to different concentrations of chloroquine and different exposure times to the drug (Table 3). It is clear that the chloroquine-associated reduction in transduction efficiency was proportional to the time of exposure to drug. To further explore this effect, chloroquine was added to cells 3 hours after transduction. The later addition of the drug improved transduction efficiency, but did not correct it completely (Table 4).

The question remains, is the effect of chloroquine due to the toxicity of drug on gene expression (it affects protein synthesis in the cells) or does it affect entry of the virus into the cell, or viral replication (reverse transcription) and integration of the vector?

To answer this question, transduced cells were cultured for 4 days (instead of the usual 2 days) before reading the FACS results to eliminate temporary toxicity to the cells. In this experiment, another subculture of the Hela cells was used, in which the transduction efficiency was lower than in previously described experiments (Table 5). Also, Hela cells were transduced with pH 5.8 growth medium, in the presence or absence of chloroquine added before transduction, to discriminate between pH-induced vector entry block from other possible effects (Table 6). The change in pH had no effect on transduction efficiency under these conditions.

Baboon CD34+ cells tolerated different concentrations of chloroquine for less than 12 hours of exposure, but longer periods caused all of the cells to die. Transduction efficiency varied in different experiments (Table 7) and although, chloroquine increased transduction in one experiment, it did not increase it in another. In these experiments, we transduced column-selected baboon bone marrow CD34+ cells four times. The transduction efficiencies without chloroquine were similar in three experiments and although, addition of 50 mmol chloroquine had no major effects in the second and third experiments, it increased transduction efficiency in the first experiment. Similarly, chloroquine at 100 mmol reduced transduction efficiency in the second and third experiments and increased it in the first one. Thus, it was concluded that chloroquine at 50 mmol had no negative effect on transduction of baboon CD34+ cells. In the second experiment, CD34+ cells were transduced by one exposure to the vector only, with and without chloroquine. Chloroquine at 50 mmol had a small negative effect on transduction efficiency (11.74 ± 0.8 without chloroquine vs 7.08 ± 0.6 at 50 mmol chloroquine). The higher chloroquine concentration (100 mmol) prevented transduction profoundly (1.31 ± 0.5).

The population of the cells in the three studies differed from each other based on lineage negativity and the number of the CD34-cell contamination was higher in the third experiment (Table 8).

The different responses to chloroquine observed during our experiments on baboon CD34+ cells may be due to different proportions of lin-positive cells (76.8% in sample 1, 24.71% in sample 2 ; undetermined for sample 3) and CD34+ cells (92.13% in sample 1, 94.63 % in sample 2 and 79.64% in sample 3).

Also, the proportion of mature large cells (according to forescatter and sidescatter character-ristic of the cells) in these three experments differed after 7 days of culture (15.38%, 12.93% and 27.85% in samples 1 – 3, respectively).

What are the exact biologic effects of chloroquine during transduction of cells with retroviral vectors?

The effects of chloroquine on CD34+ cells were more complex than other cell lines. This complexity may have been due to heterogeneity of the cell populations or a more complex process of transduction of the cells.

Also we know that chloroquine is extruded from cells by a multiple drug resistance protein (MRP) gene, so it may be possible that the concentration of this drug that is achieved inside of CD34+ cells is much lower than other cell lines.¹⁴

This study was limited by the proportion of more mature cells after transduction and also the amount of CD34 cells.

Further, the amount of PI-positive cells in these three studies was variable, and increased after chloroquine exposure. Although PI positivity in nontransduced cells varied among cells, the response of these cells to chloroquine was opposite to their response to transduction, so when chloroquine induced PI positivity, it decreased transduction efficiency and vice versa. So it may be the lower concentration of chloroquine or its use in MRP-positive cells that increases transduction, by inhibition of lysosomal enzymes and prevention of intracellular destruction of the viral particles by these enzymes.