Indian Journal of Experimental Biology Vol. 41, December 2003, pp. 1392-1399

Alterations in radiation induced cell cycle perturbations by 2-deoxy-D-glucose in human tumor cell lines

J S Adhikari l, 8 S Dwarakanath 1< Rohi l Mathuru & T Ravindranath l

I Divis ion of Biocybernetics and Radiopharmaceu tiea ls, Institute of Nuclear Medicine and Allied Scienccs, Timarpur, Delhi 110054, India

2Ambedkar Center for Biomedica l Research , Univers ity of Delhi, Delhi 110007, India

Received 8 May 2003; revised 7 JIIly 2003

In the present studies, effects of glucose analogue, 2-deoxy-D-glucose (2 -DG) on radiation-induced cell cyc le perturbations wcre in vest igated in human tumor ce ll lines. In unirradialed cells, the levels of cyclin B I in G2 phase were significantly higher in bOlh the gl ioma cell lines as compared to squamous carc inoma cel ls. Upon irradiation wi th Co60

gamma-rays (2 Gy), the cyclin B I levels were red uced in U87 cells, whi le no signi fica nt changes could be observed in other cell lines. wh ich correlated we ll with the transient G2 delay observed under these conditi ons by the BrdU pu lse chase measurements. 2-DG (5 IllM, 2 hI') induced accullluintion or cells in the G~ phase and a time-dependent increase in the levels of cyclin B I in both the glioma cell lines, while signifi ca nt changes could not be observed in any of the squamous carcinoma ce ll lines. 2-DG enhanced the cyc lin B I level fu rthcr in all the cell lines following irradiation, albeit to different ex tents. Intcres tingly, an increase in the unscheduled express ion of B I levels in G 1 phase 48 hI' after irradiation was observed in all the cel l lines investigated. 2-DG also increased the levels of cyclin DI at 24 hI' in BMG-I cell line. These observations imply that 2-DG-induccd alterations in the ce ll cyc le progression are partly responsible for its radioillodifying effects.

Keywords: Ce ll cyc le, Cyc lin. 2-Dcoxy-D-g lucosc, G2/M checkpoi nt , Radiolllodi ri cution

irradiation of eukaryotic cell results in a close-related delayed progress ion through G), S, and G2 phases of cell cyc lel.2. These delays arise on acco unt of the presence of complex cell su rveillance mechani sms, referred to as checkpoi nts, that operate mainly in response to a variety of DNA damages, although non­DNA damages have also been impl icatedJ

. At low and moderatel y absorbed doses, G ) phase arrest occ urs predominantly in cel ls with wi ld Lype p53 fu nction while G} phase de lay occurs regard less of the pS3 statu s1

.4 . The de lay in G1 phase of the ce ll cycle presumably all ows the repair and recovery processes leading to cel l survivarl

-c" while lack or abol ition of

the delay has been correlated with enhanced cell death 7

.s. Stud ies have shown that yeast cel ls with a

mutation in the rac.l 9 gene are hi ghly sens itive to radiation and do not undergo a G1 delay, thu suggesting that duration of G2 delay plays a sign ificant role in determini ng the radiosens iti vity of cell s in a dose-dependent manner6.'!.lo. T he position of cells in cel l cycle phases at the time of irradiat ion also markedly influences the extent of division dclay with

"'Corrcspondelll aut llor Fax: (+91 ) J 1-391-9509 E-mail: bsd @inlllas.org

irradiation in G 1 phase show ing min imal delay, while irradiation in Sand G2 is associated with progressive ly grea ter delaysl. Studies on transformed cell li nes predominantly show a G2 delay , even at low radi ation doses , while delay in S phase has been observed at relatively high doses tl .I? Although the mechanisms of these delays arc not completely understood, the extent of the delay appears to be

d . I IJ un er genetIC contro .

Mammalian ce ll cyc le is regulated by a set of co mplex mechani sms in volving several cycl ins and cyclin-dcpendent kinases result ing in a cascade of phosphory lat ion-dephosphorylation or target proteins directly in vo lved in DNA syn thes is, ch romosome segregation and mito ti c divisionsl4. These Cycli n­CDK complexes are influenced by a number of regul atory proteins whose levels are regul ated both by growth factor sti mul ation as well as damage leve ls through d iffere nt kin uses, p53, p21 and several other gene prod ucts l3.

Residual D A damage, the resu ltant of a competition between processes of rcpair and fixat ion of primary DNA lesions appears to be the primary cause of radiati on-induced divis ion delayl5 . Our earli er studies showed that the glucose analog,

ADHlKARl et al. : 2-00-INDUCED PERTURBATIONS IN CELL LINES 1393

2-deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolysis selectively inhibit the DNA repair and cellular recovery processes, resulting in an enhancement of the radiation damage in cells with high rates of glycolysis (like in cancer cells)'6-2'.

Under these conditions a good correlation between inhibition of DNA repair, enhanced cytogenetic damage and cell death has been demonstrated in certain cell lines,s.22, while a lack of correlation has also been observed in few cases23. Although alterations in cell cycle delay have been indicated in these studies, a clear understanding of the nature of delays as well as mechanisms involved, besides its role in the radiomodification have not emerged so far. Therefore, we have initiated systematic studies to in vestigate in detail, the cell cycle perturbations caused by 2-DG following irradiation by characterizing the nature of delays as well as alterations in some of the cell cycle regulatory proteins like cyclin B 1, D I etc. Results of these studies carried out in human glioma and squamou s carcinoma cell lines are presented here.

Materials and Methods Cell culture - Four human tumor cell lines, two

gliomas (BMG- I, U87 wild type p53) and two squamous carcinomas (4 197 wild type p53 , 4451 mutated p53) were used in these studies. Cell s were maintained in Dulbecco's modified Eagles medium (DMEM) supplemented with either 5% fetal calf serum (BMG-I) or 10% feta l cal f serum (4451 and U87) or 20% fetal calf serum (4 197), HEPES and antibio tics as described earlier'7. All experiments were carried out in exponentia ll y grow ing cells.

Treatment procedure - Cells were plated at a density of 6,000 to 8,000 cells/cm2 in petridishes. Twenty-four hours later, cells were washed with HBSS and incubated in HBSS with 5 mM 2-DG (equ imolar conc. with g lucose). Cells were irradi ated immediately following 2- DG addition and held in HBSS for 2 hr following iITadiation . Appropriate contro l groups (sham irrad iated, 2-DG and rad iation alone) were always included in the protocol. Irrad iation was carri ed out at a dose rate of 1.4 to 2 Gy per min (2 Gy) at room temperature using a cobalt-60 teletherapy unit (Eldorado, Canada).

Cell growth and cell cycle distriburio/J.- Fo ll ow­ing different treatments as described above, cells were harvested at every 24 hI' time interval. Both floating and attached ce ll s were cou nted and fixed in 70%­chilled eth anol for the analys is of cell cycle

distribution. Cell proliferation was calculated by computing the increase in cell number and an index of proliferation P, was calculated as: P = N/No where, N!= number of cells at time t;. No= number of ceUs at the time of irradiation .

Flow cytometric measurements of cellular DNA content were performed with ethanol fixed cells using the intercalating DNA fluorochrome, propidium iodide (PI) as described earlier24 . Measurements were made with a LASER based (488 nm) flowcytometer (Facs Calibur; Becton Dickinson, (USA), and data were analysed using Mod-Fit software (Becton Dickinson; USA).

Analysis of S-phase cells labeled with bromodeoxyuridine (BrdU)-Cells pulsed with 10 ~ BrdU for 30 min were trypsinized, fixed in 80% chilled ethanol and stored at 4°C. Immunostaining was performed as described earlier25

• Briefly, cells were incubated with pepsin for 10 min to isolate nuclei, and treated with 2N HCI for 30 min to partly denature DNA. Subsequently they were labeled with mouse anti-BrdU antibody (l :40, Becton Dickenson), followed by FlTC labeled anti-mouse IgG , secondary antibody (l: 100, Sigma Chemicals). The DNA was

stained with propidium iodide (PI, 50 )1g/ml) . Green (FITC) and red (PI) fluorescence were recorded for 10,000 events using cell quest software (Becton Dickinson , USA) . Data were analyzed by applying appropri ate gates fo r labeled and unlabeled cell s.

Measurement of" cyclills- Immunostaining was performed as described earlier26

. Brie fly , ethanol fixed cells was first permeabili zed with 0.25 % Triton­X 100 in PBS for 5 min on ice. Cells were then washed with PBS and incubated with respective primary antibodies di lu ted ( I :400) in PBS containing 1 % BSA, (Pharmingen, a Becton Dickinso n Co.) overn ight at 4°C. Cells were then washed with PBS containing 1 % BSA and incubated with FlTC labeled goat anti-mouse JgG, secondary antibody (S igma chemicals) diluted l: 1 00 in PBS containing 1 % BSA for 30 min at 4°C. After washi ng, cel ls were treated with 0 .1 % RNase and propidium iodide (PI, 10 I1g/ml) for 30 min. Green (FlTC) and red (PI) fluorescence were recorded for atl east 10,000 events as descri bed ea rl ier. Cell cycle was also ana lyzed from the measurement of DNA content from PI fluorescence.

Results Cell growth and cell cycle progressioll - A

significant reduction in ce ll number was observed upon treatment with 2-DG in squamous cell

1394 INDIAN J EXP BIOL, DECEMBER 2003

carcinomas (4197 and 4451). A further reduction was observed when cells were treated with 2-DG (5 mM, 2 hr) and gamma radiation (2 Gy). However, no significant effects were observed in the growth of BMG-1 and U87 cell lines (Fig. 1). Cell cycle analysis showed that both the duration and extent of G2 delay differ among these cell lines investigated (Fig. 3b). A predominant G2 delay observed at 4 hr was nearly equal in both the glioma cell lines (BMG-1 and U87) but higher in 4197 and 4451 cells (Fig. 3b).

Pulse labeling and follow up studies -Incorpora­tion of BrdU by the DNA synthesizing cells serves as a good marker for studying the progress ion of cells through cell cycle. Pul se labeling (20 min) of BMG-1 cells with BrdU at different post irradiation time intervals showed a transient delay in G2 phase of the cell cycle (data not shown). When cells were chased by monitoring the label S phase cells at various time intervals, a similar distribution of cells was observed in unirradiated cells as in pulse labeling experiment with nearly 35 % of the cells in S-phase. A significant delay in the cell cycle progression induced by 2-DG was evident particularly in irradiated cells (Fig. 2).

At 12 hr post-irradiation, nearly 41 % of the labeled cells treated with 2-DG were still in G2 phase, while this value was 34% in untreated and 36% in gamma­ray irradiated cell s. However, by 24 hr, the unirradiated cells appeared to have overcome the 2-DG-induced block, with only 20% of the labeled cells in G2 phase with a simultaneous increase in the fraction of labeled cells in G 1 phase from 11 % at 12 hr to 27% at 24 hr. Similar observations were also made in the irradiated cells treated with 2-DG, where a release of cells from G2 block was evident with nearly 10 % of the labeled cells in G2 phase and a

10.-------------------~ -a- Control .... 2 Gy ~2-DG ..... 2-DG+2 Gy (a)

concomitant increase in the fraction of labeled cells in G 1 phase from 7% at 12 hr to 35% at 24 hr.

Cyciins-Constitutive as well as treatment­induced changes in cyclin B 1 levels were measured in all cell lines by imrnunoflowcytometry. The constitutive levels of cyclin B 1 were higher in both the glioma cell lines compared to squamous carcinoma cell lines (Fig. 3a). However, treatment­induced alterations in the cyclin B 1 levels varied considerably among the different cell lines. Upon irradiation (2 Gy), the cyclin B 1 levels were reduced in U87 cells at 24 hr post-irradiation, while no significant change could be observed in other cell lines. However, in unirradi ated cells, presence of 2-DG for 2 hr enhanced the level of cyclin B 1 in all the cell lines investigated, which was elevated further in irradiated cells at 24 hr (Fig. 3a).

Since events responsible for disturbances in cell cycle progression are linked to the radiation-induced damage and competes with DNA repair processes immediately following the induction of damage, the effects of 2-DG on ceJl cycle kinetics were studied at short time intervals up to 2 hr following irradiation. Cyelin B 1 levels were significantly higher at 4 hr but reduced subsequently by 12 hr fo llowing the treatment in BMG-l cells (Fig. 4a), indicating a transient disturbance by this short exposure to 2-DG. The extent of this increase in cyclin B 1 levels were more in both the glioma cells as compared to carcinoma cells to 2-DG treatment (data not shown). Although, the cyelin B 1 levels were observed higher in G2 phase, where it plays a role in facilitating G2-M transition, interestingly a significant unscheduled expression was observed in the G 1 phase in all the cell lines after 48 hr of treatment (Fig . 4a).

10 I (b)

I

0.1 OL---- 2-4----4-S----7-2 ---'so 0.10 24 4S 72 so

Time (hr)

Fig. 1 - Cell generation curves from a typical experiment showing the effects of gamma rays (absorbed dose-2 Gy) and 2-DG (5 mM, 2 hr) on the growth of log phase BMG-I (a) and 4197 (b) cell lines.

ADHIKARI et al.: 2-DG-INDUCED PERTURBATIONS IN CELL LINES 1395

Since 2-DG was also found to induce disturbances in the progression of cells from G 1 to S phase, we investigated the levels of cyclin Dl in BMG- l cells under these conditions. 2-DG significantly increased cyc1in D 1 levels in un irradiated and irradiated cells at 24 hr post treatment, while no significant change could be observed further at later time intervals (Fig. 5) .

Discussion Primary lesions (both DNA as well as non DNA)

caused by radiation initiate a set of competitive cellular responses such as DNA repair, cell cycle peturbations, disturbances in signal transduction pathways and apoptosis, involving complex molecular

" o

-o

J o

Control 2Gy

processes driven by various kinases, phosphatases and polymerases. Some of which are highly regulated by flow of metabolic energy. Therefore, modification of cellular radiation response by metaboli c inhibitors such as 2-DG (and others like 6AN, hematoporphyrin derivative) needs to be understood from their integrated effects on these processes. Residual DNA damage, resultant of a competition between DNA repair and damage fixation processes is one of the

. d . f II I d·· 27 28 main etermlnants 0 ce . u ar ra latlOn response . . Present studies indicate that 2-DG-induced alterations in the progression of cells through different phases of cell cycle occurs through DNA damage mediated checkpoint arrest, which vary among different cell

2-DG 2-DG+2Gy

01022k2 .014

"';':,

o hr

4 hr

12 hr

24 hr

200 400

Relative DNA Content

Fig. 2 - Bivariate plots of DNA and anti -BrdU anti body levels showing the effects of 2-DG (S 111M, 2 hr) on radiation-induced cell cycle perturbations analyzed by chasing BrdU labeled exponentially growing BMG- \ cells.

1396 INDIAN J EXP BIOL, DECEMBER 2003

types. DNA damage has been implicated as the primary signal responsible for the operation of cell cycle checkpoints at G1/S and G2/M transition although , recent evidences indicate that non DNA damage could also playa role3

. Heterogeneity in the alterations in cell cycle progression observed here (Fig. 4 b) may be partly responsible fo r the observed differences in the survival by 2-DG and could arise on account of variations ID the effects of 2-DG on

Control 2 Gy

Relative DNA Content

(b) Control 2 Gy ·2-DG 2-DG + 2 Gy

Relative DNA Content

Fig. 3 - I III Illuno-I'low cytomctric measurement showing effects of gamma rays (2 Gy) and 2-DG (5 mi\ll. 2 hI') on cyc lin B 1 level s in exponclllially growing human tumor cel l lines, observed 24 hI' after treatments. (a) Bivariate plots of cyclin B I and re lat ive DNA content; and (b) Content showing the treatmelll induced distur­bances in the cell cycle progression.

processes of DNA repair, induction of p21 -mediated apoptosis or DNA damage-dependent p53-mediated progression in cell cycle.

2-DG-induced energy linked differences in strand break rejoining among human tumor cell Jines have been reported25 While no significant di fferences in the residual DNA damage were observed upon treatment with 2-DG in a squamolls carcinoma cell line (4 197), the residual D A damage under sintilar conditions was signifi cantly higher in both the glioma cell li nes (BMG-l and U87) and in another squamous carcinoma cell line (445 1)23. Presence of 2-DG for 2 hr following irrad iation resulted in a higher growth inhibition in 4197 cell s than 4451 cells (Fig . 1) while,

Rela tive DNA contcnt

Fig. 4-Bivariata plots of cycli n Bl and DNA content showing kinetics of cyclin B I levels observed upon treatment with gamma rays (2 Gy) and 2-DG (5 mM, 2 hr) in (a) exponentia ll y growing BMG- I cells . (b) Histogram plots of relative DNA contents show­ing treatment induced disturbances in cell cycle progression.

ADHlKARI el at.: 2-DG-INDUCED PERTURBATIONS IN CELL LINES 1397

no significant inhibition of growth was observed in both the glioma cell lines (BMG-1 and U87). 2-DG (5 mM) induced delay in cell cycle progression was more pronounced in the G2 to M phase transition. While delay in the transition of G 1 phase cells into the S-phase was evident, no correlation was observed in residual DNA damage and cell survival, therefore the role of DNA damage-dependent p53-mediated cell cycle progress ion was speculated. However, we observed that 4197, BMG- l and U87 carrying wt p53 differ in their sensitivity to 2-0G treatment (Fig. 1). Thus, it appears that neither residual damage nor p53 status is sole determinant of 2-0G-induced cell cycle perturbations as reported earlier4

,29.

A direct effect of transient alterations in the metabolic disturbances caused by 2-0G leading to alterations in some of the cell cycle regulatory processes particularly involving the maintenance of appropriate cyclins is indicated by the present studies. Small, yet significant changes in the cyclin Bland D1 levels observed in unirradiated BMG-1 cells, 24-72 hr after a short exposure (2 hr) to 2-0G is an indication of such a possibility (Fig. 4a). The transient increase in the levels of cyclin Blat 4 hr in both unilTadiated and irradiated cells treated with 2-0G correlates well with the delayed progression from G2 to M phase of cell cycle (Fig. 4b). Earlier studies have indeed shown a correlation between fall in A TP levels and accumu­lation of cells in G2 and G 1 phases of the cell cycle and concentration-dependent growth inhibition22

,24.32. BrdU pulse chase experiments have clearly shown that the treatment of cells with 2-0G enhanced the radiation­induced G2 delay later at 4 hr (Fig. 2). It is interesting to note that in both squamous carcinoma cell lines where radiosensitization by 2-DG was not observed, the constitutive cyclin B 1 levels were significantly

Control. 2Gv

~ ~ I ~["I~ "~ I u ~ ': ~ - ) ... _",\.--r .. :::;; -.0.- 0

lower as compared to the glioma cells (Fig. 3a). Therefore, 2-DG-induced radiosensitization in U87 and BMG-l cells (both wt p53) appears to be partly related to the constitutive as well as treatment-induced alterations in cyclin B 1 levels. One of the reasons could be the damage-dependent cyclin Bl-mediated enhanced cell death through apoptosis, as has been reported in cells with higher level of cyclin B 131.

Indeed, the radiation-induced delayed apoptosis was higher in both glioma cell lines as compared to the sqamous carcinoma cell lines32. Since the role of cyclin Olin radiosensitization has been indicated earlier33

, we investigated the changes in cyclin 01 levels in altered cell cycle progression upon treatment with 2-DG. Further, enhanced levels of cyclin D I observed 24 hr after treatment with 2-0G alone or in combination with radiation (Fig. 5) indicates another pathway possibly modulated by 2-DG in the regulation of cell cycle progression. These responses could , however, vary among cell lines as shown here between the two glioma and squamous carcinoma cell lines, which could partly contribute to heterogeneity in the radiosensitization by, 2-0G.

Taken together, the results of the present studies suggest that presence of 2-0G for few hours following irradiation significantly alters the progression of cells through the cell cycle, which may be partly responsible for 2-DG-induced alterations in cellular response observed earlier24

.25

,32. Quantitatively, these alterations were different in different cell lines, in line with the heterogeneity in the overall radiomodification by 2-DG observed in these cells23

.

Although changes in cyclin B 1 level are implicated to be responsible for alterations in cell cycle progression, role of transient changes in energy levels, changes in levels of cyclin D1 which manifest at earlier time

2-0G 2-DG+2Gy

Relative DNA content

Fig. 5 - Immunol1owcytometric measurements of cyclin DJ levels in exponentially growing BMG-I cells, 24 hr after treatment with gamma rays (2 Gy) and 2-DG (5 mM, 2 hr). Lower panel indicates DNA hi stograms of respective groups

1398 INDIAN J EXP BIOL, DECEMBER 2003

intervals upon short duration treatment of 2-DG could also play important roles. Further studies are required to establish a causal relationship between energy status and cyclin levels as well as their roles in radiomodification by 2-DG.

Acknowledgement We kindly acknowledge the technical support

provided by Miss Divya Khaitan fo r maintaining the cell lines. Useful discussions with Dr. Rajeev Varshney, Dr. Seema Gupta and Shailja Singh are acknowledged. These studies have been supported by a DRDO project (lNM-280), Ministry of Defence, Govt. of Indi a. Mr. Rohit Mathur is a recipient of junior research fellowship from CSIR.

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