Jpn. J. Protozool. Vol. 37, No. 2. (2004)

127

Original
Analysis of a factor released from UV light-irradiated Dictyostelium
discoideum
Hiro YASUKAWA
Division of Bioengineering, Faculty of Engineering,
Toyama University, Toyama 930-8555, Japan.

SUMMARY

INTRODUCTION

The behavior of the social amoeba Dictyostelium discoideum following UV irradiation wasanalyzed. Cells cultured in a liquid medium migrated at 4.5 + 0.7 µm/min, while their motility
increased to 19.4 + 1.7 µm/min following UV irradiation (50 J/m2). A conditioned medium preparedfrom the irradiated cells, CM (UV), was used in
the following experiments. Motility of cells that
had not been exposed to UV increased to 29.7 +2.8 µm/min when the medium was changed to CM(UV). In addition, the expression levels of recA
and rad51 were increased and sensitivity to UV
was decreased in these cells. These results indicate that UV light-irradiated D. discoideum cells
release a factor that enhances motility and induces
expression of DNA repair enzymes in unirradiatedcells. The factor would be important for D. discoideum to alleviate damage.

It is important for organisms to maintaintheir genomic integrity. However, they are continuously exposed to various DNA-damaging
agents such as UV light, ionizing radiation andchemicals. Organisms therefore have mechanisms
by which DNA damage is identified and repaired.
The social amoeba Dictyostelium discoideum, a lower eukaryote feeding on bacteria,
has been shown to be resistant to DNA-damaging
agents (Deering, 1988; Yu et al., 1998). Several
genes responsible for DNA repair have been foundand characterized in this organism. APNA, formerly called DdAPN, and APEA are AP endonucleases (Freeland et al., 1996; Tsuji et al., 2001).
repB, repD and repE encode the homologs of thehuman XPB DNA helicase, XPD DNA helicase
and the damage recognition molecule UVDDB/XPE, respectively (Alexander et al., 1996; Lee etal., 1997). A Rad51 homolog has been found in
the database (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html). Recently, a RecA homolog
localizing in mitochondria has been identified andcharacterized (Hasegawa et al., 2004). Lesions in
the D. discoideum genome are repaired by theseenzymes.

Tel & Fax: 81 76 445 6875
E-mail: hiro@eng.toyama-u.ac.jp
Received: 23 Aug 2004; Accepted: 8 Nov 2004.

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A signal from UV irradiated D. discoideum

It is also important for organisms to takerefuge when they are exposed to DNA-damaging
agents. If there are agents nearby the organisms,
the organisms should escape from them as soon as
possible to avoid lethal damage. Experimental
result demonstrating that D. discoideum takes refuge when exposed to UV light is shown in thisreport. In addition, the experimental evidence
showing here demonstrates that UV lightirradiated D. discoideum released a factor that increased motility and induced DNA repair enzymes
in unirradiated cells.

MATERIALS AND METHODS
Cell line and culture condition
D. discoideum Ax2 was used in the experiments. Cells were cultured in HL5 medium (0.04%KH2PO4, 0.05% Na2HPO4, 0.7% yeast extract,
1.4% bacteriological peptone and 1% glucose, pH
6.5).
Motility assay of UV light-irradiated cells
D. discoideum cells were placed with 20 µl
HL5 medium on a glass slide at a density of 2.0 ×
104 cells/cm2. After 20 min, the medium was removed from the glass slide. The cells on the slidewere irradiated by 50-J/m2 UV light (254 nm) by
the use of a UV crosslinker CL-1000 (UVP, CA,USA) and then incubated with a fresh HL5 medium (20 µl). Cells on the slide were photographed at 2-min intervals (~20 min) to determine
migration speed. For the control, cells were placedwith 20 µl HL5 medium on a slide at a density of2.0 × 104 cells/cm2. After 20 min, the medium waschanged to 20 µl fresh HL5 medium. These cells
were also photographed at 2-min intervals to determine the speed. Some of the cells did not migrate
either in control or exposed fractions. Cell motility was determined without these cells.

Motility assay in conditioned medium
Preparation of conditioned medium: Cells
were placed with 20 µl HL5 medium on a glass
slide at a density of 2.0 × 104 cells/cm2 for 20 min.
The medium was changed to 20 µl fresh HL5 medium. After 20 min, the medium was removed
from the slide and used as a control conditioned
medium, CM. Preparation of conditioned mediumof UV light-irradiated cells: Cells were placed with20 µl HL5 medium on a glass slide at a density of
2.0 × 104 cells/cm2. After 20 min, the medium wasremoved from the slide. The cells on the slidewere irradiated by 50-J/m2 UV light and then incubated with 20 µl fresh HL5 medium for 20 min.
The medium in which UV light-irradiated cells
were incubated, CM(UV), was recovered. Assays:Cells were placed with HL5 medium on two slides(2.0 × 104 cells/cm2). HL5 medium on the slides
was replaced by CM or CM(UV). Cells were photographed at 2-min intervals (~20 min) to determine migration speed.
Gene expression analysis
Preparation of conditioned medium: Cells
were cultured with 10 ml HL5 medium in a 90-mm
dish until the density reached 6.0 × 104 cells/cm2.
Medium in the dish was removed, and the cells
were rinsed with a phosphate buffer (1.66 g NaH2PO4 in 1 L H2O, pH adjusted to 6.1 with KOH).
Cells were then incubated with 10 ml fresh HL5medium for 20 min. The medium was removed
from the dish and used as a control conditioned
medium, CM. Preparation of conditioned medium
of UV light-irradiated cells: Cells were cultured
with 10 ml HL5 medium in a 90-mm dish until thedensity reached 6.0 × 104 cells/cm2. The cells inthe dish were rinsed with the phosphate buffer,irradiated by 50-J/m2 UV light, and then incubatedwith 10 ml fresh HL5 medium for 20 min. The
medium in which UV light-irradiated cells werecultured, CM(UV), was recovered. Assays: Cells
were cultured with 10 ml HL5 medium in twoJpn. J. Protozool. Vol. 37, No. 2. (2004)

129

Fig. 1. Motility of cells exposed to UV light. Unexposed cells (control cells) and UV light-irradiatedcells were photographed at 2-min intervals to determine migration speed. Migration speed (mean +S.E.) was determined from 29 cells (control cells)and 41 cells (UV light-irradiated cells) obtained
from two independent experiments.

Fig. 2. Motility of cells in the conditioned medium.
Cell migration speed in CM and CM(UV) wasanalyzed. Migration speed (mean + S.E.) was
measured from 29 cells in CM and 21 cells in CM(UV) obtained from two independent experiments.

dishes (6.0 x 104 cells/cm2). HL5 medium in thedishes was replaced by CM or CM(UV). After 40
min, cells were harvested for RNA preparation.
Expression levels of recA and rad51 were analyzed by RT-PCR by the use of a One Step RTPCR Kit (Takara Shuzo, Kyoto, Japan). The primers specific for rad51 were 5'-GATGGTGGTATTGAATCTGGTTCAATACTG and 5'-GACGGCAACACCAAACTCATCGGCTAATCT. The nucleotide sequences of primers for recA and actin15are described in previous reports (Akaza et al.,

2002; Hasegawa et al., 2004). DNA fragments
were amplified 14-24 cycles after reverse transcription and run through an agarose gel to compare signal intensities.
UV light sensitivity assay
CM and CM(UV) were prepared from dishcultures as described above. Equal numbers of D.
discoideum cells were incubated with CM or CM(UV) for 40 min. Cells were then washed with thephosphate buffer and irradiated by 200-J/m2 UV

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A signal from UV irradiated D. discoideumCMCM (UV)
recA

rad51
actin15

Fig. 3. Expression of recA and rad51. Expression
levels of recA and rad51 were analyzed by RT-PCR
as described in Materials and Methods. The reaction
products of recA (18 cycles), rad51 (16 cycles) andactin15 (14 cycles) are presented.

Fig. 4. Dead cells in CM or CM(UV). Cells
incubated with CM and CM(UV) were exposed
to UV light as described in Materials andMethods. Dead cells that were stained byphloxine B and viable cells were counted undera microscope. Three sets of experiments werecarried out.

light. Then the cells were incubated with a freshHL5 medium for 20 h. Dead cells that werestained by phloxine B and viable cells werecounted under a microscope as described in a previous report (Garcia et al., 2000).

RESULTS AND DISCUSSION
D. discoideum exposed to UV light increased
motility
Experiments were carried out to examine
whether D. discoideum would increase motility
when exposed to UV light. D. discoideum cells
were placed on a glass slide with a portion of HL5medium for 20 min. The liquid medium was removed and the cells were exposed to UV light (50J/m2). Irradiation of 50 J/m2 is a sublethal condi-tion for D. discoideum (Garcia et al., 2000; Hasegawa et al., 2004). Soon after the irradiation, afresh medium was placed on the cells. Unirradiated cells migrated at 4.5 + 0.7 µm/min, while UV
irradiated cells migrated at 19.4 + 1.7 µm/min(Fig. 1). The p value obtained from a t test wasless than 0.01. The results indicate that UV irradiation increased the motility of the cells. Increasein motility would be important for D. discoideum
to take refuge from DNA-damaging agents.
UV light-irradiated D. discoideum released afactor which increased motility in unirradiatedcells
Refuge from a damaging agent prior to exposure would be of great benefit for organisms
because organisms that have escaped prior to exposure to an agent are able to survive and grow at

Jpn. J. Protozool. Vol. 37, No. 2. (2004)

a place of refuge. Exposed organisms might therefore release a signal to increase motility in unexposed organisms. Unexposed organisms would beable to escape from the agent and survive at theexpense of exposed organisms. To determine
whether D. discoideum releases such a signal, thefollowing experiments were carried out. A medium in which UV light-irradiated cells had been
incubated for 20 min was removed and used as aconditioned medium, CM (UV). Cells that had notbeen exposed to UV light were incubatedwith the CM (UV) and observed under a microscope. The migration speed of these cells increased
remarkably (29.7 + 2.8 µm/min) even though they
had not been exposed to UV light (Fig. 2). For acontrol, a medium in which unexposed cells wereincubated for 20 min was removed as a conditioned medium, CM. Migration speed of cells in
the CM was 6.2 + 0.7 µm/min (Fig. 2). The results of a t test (p > 0.05) showed that this value
was not different from the value obtained from thecells in HL5 medium (4.5 + 0.7 µm/min). These
results indicate that a factor that significantly enhanced motility was released from UV lightirradiated D. discoideum but not from unirradiatedcells.
Released factor decreased UV light sensitivityof D. discoideum
It has been observed that D. discoideum
increases expression of recA (by 16 fold) andrad51 (by 10 fold) following exposure to 50-J/m2
UV irradiation (Hasegawa et al., 2004 and unpublished result). The following experiment was carried out to examine whether the factor in CM(UV)
would induce expression of these genes in unirradiated cells. D. discoideum cells incubated withCM or CM(UV) were collected to extract total
RNA for RT-PCR analysis. The RT-PCR products
were resolved by gel electrophoresis and their signal intensities were analyzed. Expression ofactin15 was analyzed as an experimental control as

131

described in previous reports (Akaza et al., 2002;
Hasegawa et al., 2004). Expression levels of recA
and rad51 in the cells in CM(UV) were higher(~3-fold) than those in the cells in CM. Theseresults indicate that a factor in CM(UV) induced
DNA repair enzymes in cells even though the cells
were not exposed to UV light (Fig. 3).
The next experiment was performed to determine whether the induction of DNA repair enzymes contributes to the decrease in UV sensitivityof D. discoideum. Equal numbers of cells wereincubated with CM or CM(UV) for 40 min, irradiated by 200-J/m2 UV light and then incubated witha fresh HL5 medium for 20 h. Dead cells werecounted following dye staining. Relative numberof dead cells is shown with the result obtained
from a t test (Fig. 4). The UV light sensitivity ofD. discoideum that had been incubated with CM(UV) decreased (by 0.49 + 0.07 fold) comparedwith that of the cells that had been incubated withCM.
The experimental evidence presented here
demonstrates that UV light-irradiated D. discoideum released a factor that increased motilityand induced DNA repair enzymes in unirradiatedcells. This factor would be important for D. discoideum to alleviate damage and avoid annihilation in an emergency. Molecular identification ofthe factor is in progress.
ACKNOWLEDGMENT
The author wishes to thank Y.Hasegawa(Toyama University) for helpful support.

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A signal from UV irradiated D. discoideum

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