VanWijk et al (1993)-Light-induced photon emission by mammalian cells.pdf

7/28/2019 VanWijk et al (1993)-Light-induced photon emission by mammalian cells.pdf

1/5

J. Photochem. Photobiol. B : Biol., 18 (1993) 75-79 75

Light-induced photon emission by mammalian cellsRoeland van Wijk*+, Hans van Akerf, Weiping Meib and Fritz A. PoppbDepartment of Molecular Cell B iology, State University, Padualaan 8, 3584 CH Utr echt (Netherlands)bInstitu te of Biophysics, Technology Centre, Opelstr asse IO, 6750 Kati erslautern 25 (Getmany)(Received July 31, 1992; accepted October 27, 1992)

AbstractIn this work, the light-induced photon emission (IPE) by suspensions of mammalian cells was examined. IPE isextremely low and for detection a single photon counting device with a cooled EM1 9558QB photomultipliertube was used. The mammalian cells in this study were from different tissues and different mammalian speciesincluding cat, Chinese hamster, cow, dog, human, monkey, mouse and rat. The IPE was detected in all mammaliancells tested, but was different for the various cell types, ranging from 4 to 100 photons per lo4 cells. Althoughour data agree with previous studies in that the IPE of non-fibroblastic normal cells is distinct from that ofmalignant cells our results reveal that cells of fibroblastic origin show the highest IPE values.

Keywords: Induced photon emission, single photon counting, mammalian cells

1. IntroductionRecent studies have demonstrated re-scatteredemission by suspensions of mammalian cells after

brief illumination with an ordinary light source[l-4]. This light-induced photon emission (IPE)is extremely weak and special detectors and deviceshave been constructed for its detection [5, 61. Inthe few comparative studies on the IPE of acultured tumour cell type and its normal coun-terpart, characteristic differences were observedbetween the normal and transformed cell, yieldingthe lowest value for the normal cell and the highestvalue for the tumour cell [l-4]. However, thequantitative data of these four studies cannot becompared, since the measurement conditions weredifferent in these investigations. We have drawnup an inventory of the mammalian cell types presentin our laboratory with respect to their IPE activity.We report the IPE of cells from different tissuesand different mammalian species, including cat,Chinese hamster, cow, dog, human, monkey, mouseand rat.2. Materials and methods

In this study, primary cultures, established nor-mal ceil lines and tumour cell lines were used.Author to whom correspondence should be addressed.

loll-1344/93/$6.00

2.1. Primary culturesRat hepatocytes were isolated essentially as de-scribed previously [7]. For IPE measurementsfreshly isolated cells were used.Rat liver fibroblasts were obtained by long-termculturing of the liver cell population after platingat low density. Under these conditions rat liverfibroblasts were able to divide rapidly. After 2weeks of culture the hepatocytes represented lessthan 5% of the population, which consisted of95% fibroblasts. After two further subcultures cellswere used for measurement.Rat heart fibroblasts were obtained from heart

cell cultures [S], which were prepared accordingto a modification of the method of Harary andFarley [9]. Neonatal rats (l-2 days old) weredecapitated and the hearts were excised andminced. The mince was incubated in a spinnerflask at 37 C with 0.05%-0.1% trypsin (in 137mM NaCl, 5 mM KCl, 4 mM NaHC03, 5 mMglucose, penicillin (100 000 units 1-l) and strep-tomycin (100 mg 1-l). The incubation fluid wasdecanted and new medium was added. The su-pernatant from the first three incubations (15 mineach) was discarded; during the following 6-8incubations (10 min each), the mince was almostcompletely digested. Cell pellets were spun (8 min,43Og) and resuspended in Hams FlO growth me-dium (Gibco), supplemented with 10% foetal calf

0 1993 - Elsevier Sequoia. Ail rights reserved


2/5

76 R. van Wi jk et al. I Li ght-induced photon emission by mammalian cell s

serum, 10 mM NaHCO,, penicillin (100 000 unitsI-), streptomycin (100 mg l-l), arabinose C (10PM, to inhibit fibroblast growth) and CaCl, (finalconcentration, 1 mM). The cells were plated onFalcon 3000 dishes for 4 h, during which timehbroblasts adhere and myocytes remain freely sus-pended. Fibroblasts which remained on the surfaceof the dish were trypsinized again. They werereplated in Dulbeccos Minimal Essential Medium(DMEM, Gibco, supplemented with 10% foetalcalf serum) several times and cultured for severalweeks before use in the measurements.

(2) Reuber H35 cell line [12] and HTC cell line[13] were established from transplantable rat he-patomas.(3) The cat breast carcinoma cell line K248Cwas obtained from Dr. H. Nederbragt (Departmentof Pathology, Faculty of Veterinary Sciences, StateUniversity, Utrecht, Netherlands).(4) The human lung tumour cell line SW1573was originally isolated and characterized as a squa-mous cell carcinoma by Dr. A. Leibovitz [14].

2.4. Cell culturingHuman foreskin fibroblasts were isolated es-sentially as described previously [lo].

2.2. Normal cell li nesThe normal cell lines used in this study havebeen derived from the American Type CultureCollection [ll]. The catalogue numbers and theorigin of the individual lines are as follows.(1) CCLl: NCTC clone 929 derived from theparental strain L, which was derived from normalsubcutaneous areolar and adipose tissue of a lOO-day-old male C3H/An mouse.(2) CCL34: MDCK (NBL-2) cell line, whichwas derived from the kidney of a normal, adult,female cocker spaniel.

Normal and tumour cells, with the exception ofrat hepatocytes, were adapted to growth in DMEMsupplemented with 10% foetal calf serum, andbuffered with 10 mM N-2_hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES, pH 7.4). Cellsuspensions of monolayers of normal and tumourcells were prepared by trypsinization. After tryp-sinization the cells were washed and resuspendedin DMEM without phenol red for IPE measure-ments as described previously [3].2.5. Determination of photon emission

(3) CCL61: CHO-Kl cells derived as subclonefrom the parental CHO cell line, which was initiatedfrom a biopsy of an ovary of an adult Chinesehamster.(4) CCL81: Vero cell line, which was initiatedfrom the kidney of a normal, adult, African greenmonkey.(5) CCL92: 3T3-Swiss albino cell line, whichwas established from disaggregated Swiss mouseembryos; they are contact-sensitive fibroblasts.(6) CCL226: C3HlOTi clone 8 cells, which wereisolated from a line of C3H mouse embryo cells;they are contact-sensitive fibroblasts.(7) CRL1395: FBHE cell line, which was es-tablished from foetal bovine heart endothelial cells.(8) CRL1658: NIH/3T3 cell line of highly con-tact-sensitive cells, which was established fromNIH Swiss mouse embryo cultures in the samemanner as the 3T3 fibroblasts.2.3. Tumour cell lines

For detection and registration of IPE we usedthe single photon counting device described pre-viously [3]. This device was equipped with a cooledEM1 9558QB photomultiplier tube. The cathodehas a diameter of 44 mm and is sensitive in therange 200-800 nm. The average quantum efficiencyin this range was approximately 10%. The pho-totube output was connected to an amplifier-dis-criminator and counted by a dual counter. Thephotomultiplier was cooled to -40 C which re-duced the dark count rate to about 35 counts persecond (c.P.s.). It was further equipped with anoptical filter unit and a shutter to block the opticalpath to the sample compartment. A 2 cm quartzcuvette containing 11 ml of cell suspension ormedium was placed in the darkened sample com-partment at a distance of 125 mm from the pho-tomultiplier. In general, cell densities up to 105cells ml- were used for IPE measurements, withthe exception of some cell types which were usedat higher cell densities. The sample compartmentwas kept at 37 C. Prior to the commencementof the measurements, the samples were kept incomplete darkness for 5 min at 37 C and con-tinuously stirred.

The tumour cell lines used in this study are The photon emission intensity was measuredcharacterized as follows. and integrated over intervals of 50 ms by computer.(1) Clone Neuro-2A was established from a For stimulation of photon emission a 150 W halogenspontaneous tumour (neuroblastoma) of a strain tungsten lamp (7158, Philips, Eindhoven, Neth-A albino mouse (American Type Culture Collection erlands) was used. It was situated at right anglesCCL131) [ll]. to the photomultiplier and was equipped with a


3/5

R. van Wqk et al, / Li ght-induced photon emission by mammal ian celL FTABLE 1. Induced photon emission of mammalian cells

71

Species Cell type P/Cb IPE per lo4 cells ODSss per lo6 cellsd1. Rat2. Cow3. Human4. Mouse5. Rat6. Mouse7. Human8. Mouse9. Rat

10. Mouse11. Cat12. Rat13. Chinesehamster14. Rat15. Dog16. Mouse

17. Monkey

Liver fibroblastsBovine endothelial cells (CRL1395)Foreskin fibroblasts3T3 fibroblasts (CCL92)Heart fibroblastsC3HlOTi fibroblasts (CCL226)Lung carcinoma SW1573NIW3T3 (CRL1658)Hepatoma HTCNeuroblastoma N,A (CCL131)Breast carcinoma K248CHepatoma Reuber H35Ovary cellsHepatocytesMDCK kidney cells (CCL34)NCIC clone 929 areolar andadipose cells (CCLl)Vero kidney cells (CCL81)

NNNNNNTTTTTTNNNN

N

PCPCPCCCCCCCCPCC

C

114.2 f 29.8 1.15366.5+ 9.8 0.16764.4 f 10.8 0.51858.1i- 5.4 0.17437.2f 4.7 0.19536.4* 9.9 0.43036.3&- 4.8 0.62028.1* 5.1 0.29815.6* 1.8 0.28114.9* 4.1 0.329

8.3* 1.1 0.1447.6+ 0.6 0.1807.6+ 1.1 0.0996.1 f 0.8 0.5645.6f0.4 0.1565.5* 0.8 0.126

4.1* 1.0 0.098N/I, normal or tumour.bP/C, primary culture or cell line.Average value (from at least three experiments) and standard deviation.dODSsJ, optical density at 555 nm.

second filter unit cutting out wavelengths above720 nm and below 310 nm. The distance betweenthe lamp and the cuvette was 19 cm. No tem-perature changes were observed during the illu-mination period. During the pre-illumination theshutter was closed. Each measurement cycle wasstarted by irradiating the sample for 10 s. Theshutter was opened after closing the lamp com-partment and turning out the lamp; the emissionwas recorded (and evaluated) from 0.3 to 5.3 safter the end of illumination. Each sample wasmeasured at least three times.

3. ResultsExperiments were performed on cell suspensionsof cat, Chinese hamster, cow, dog, human, monkey,mouse and rat origin. Several of these cell sus-pensions were derived from tissues which weredisaggregated or kept as primary cultures; otherswere prepared from cell lines that had been grownin monolayer culture and were considered as es-tablished cell lines. The cell lines were either

normal, being diploid or slightly hypodiploid, or

tumour. The characteristics are summarized inTable 1.The decay behaviour of IPE from 0.3 to 5.3 sfor the mammalian cell suspensions was similarto that described previously for HTC cells [3].The decay curves for photon emission decreasedcontinuously and IPE in the period O-3-1.3 sincluded 90% of the total IPE, whereas after 5s the IPE values were similar to the dark countrate obtained without prior illumination. As ameasure of IPE intensity we calculated the totalamount of photon emission over the measurementperiod up to 1 s by the accumulation of the 50ms emission values. In preliminary experimentswe detected some IPE after irradiation of an emptycuvette or a cuvette with medium but withoutcells; selection of quartz cuvettes resulted in alow non-cellular background, which was subtractedin order to obtain the cell-specific IPE values.By expressing the total number of photon countsin 1 s as a function of cell density, the cellsdisplayed an increasing IPE with increasing celldensity (Fig. 1). The IPE per cell was differentfor the various cell types, ranging from 4 to 100photons per lo4 cells (Table 1).


4/5

78 R. van Wij k et al. / Li ght-induced photon emission by mammali an cell s2 53 6 9

14 78 10

111213

141516

17

0 1 234567Cells/ml (*l 0e6)

Fig. 1. Cell density dependence of total IPE of suspensions ofmammalian cells, after illumination with white light. The totalnumber of photons was calculated by adding the number ofphotons up to 1 s after illumination. Points represent the averageof three determinations of the IPE (error represented by verticalbar) and cell number (error represented by horizontal bar). Thenumbers outside the figure represent the different cell types andcorrespond to the numbers given in Table 1.

From these data we believe that IPE is a generalphenomenon in suspension of mammalian cells.For comparison, we evaluated the IPE as a functionof several cellular characteristics: (1) the speciesof origin; (2) the type of cell; (3) the degree oftransformation. A relationship between the IPEand the species of origin was not observed. Withinone species, especially rat and mouse, differentcell types show a large variation in IPE. Therelationship between IPE and specific cell type ismost obvious. Cells of fibroblastic origin, eitherobtained from primary cultures of dissociated cellsor derived from monolayer cultures of establishedcell lines, show the highest values. The establishedcell line of foetal bovine endothelial origin alsobelongs to the group of high IPE activity, suggestingthat a high IPE value is characteristic of mesod-ermal cells. The other cell types tested were eitherectodermal or entodermal in origin. The group ofcell types of non-mesodermal origin included nor-mal cells and tumour cells. Although in this studytumour cells have been compared with their normal

counterparts in a single case only (liver and hep-atoma cells), in general it is obvious that the IPEvalues of suspensions of normal cells are low,whereas tumour cells have IPE values rangingbetween the normal and fibroblast-type cells.Another factor which has been considered tobe related to IPE is the size of the cell. Frommicroscopic examination of the cell suspensionsit was apparent that the mammalian cells understudy differ with respect to their size. In parallelmeasurements the optical density at 555 nm ofthe cell suspensions was measured spectropho-tometrically at the densities employed in the IPEmeasurements. The optical density is directly pro-portional to the cell density under our experimentalconditions. The differences in optical density ofthe suspensions reflect differences in the cell size(Table 1). Cell types were very different withrespect to cell size, Vero kidney cells and Chinesehamster ovary cells being the smallest, and he-patocytes and liver fibroblasts being the largest.We found no relation between IPE and cell size.When IPE is expressed relative to optical density,as a measure of cell size, the large differencesbetween the IPE values of different cell typesremain.4. Discussion

The results confirm that suspensions of mam-malian cells show a weak light-induced photonemission. The intensity of this emission is depen-dent on the cell type. In general, the lowest IPEvalues ranging between 4 and 8 photons per lo4cells are found for normal, non-mesodermal, cells.The IPE values for tumour cells range between7 and 36 photons per lo4 cells. Relatively highIPE values, between 30 and 100 photons per lo4cells, are found in suspensions of fibroblasts. Re-cently, data on the spontaneous photon emissionof normal and tumour tissue have been published1151. Spontaneous photon emission is lower innormal tissue than in tumour tissue. Since differenttypes of cells, including fibroblasts, are present inisolated tissues, further research is required tocompare the light-induced with the spontaneousphoton emission of tumour and normal cells.A consistent explanation for the IPE activity ofsuspensions of mammalian cells is currently lacking.It was reported that the IPE activity was notretained in the cytoplasmic fraction after cell frac-tionation. Instead, the IPE activity was found aftertesting the fraction containing nuclei [3, 41. How-ever, extracted DNA has no IPE activity [3, 41.Further understanding of the molecular basisof IPE requires spectral analyses of the activation


5/5

R. van Wgk et al. / Li ght-induced photon emission by mammalian cells 19

of mammalian IPE and of IPE itself. This is difficultdue to the low level of photon emission afterillumination by white light. Our data allow theselection of other cell types for further analyses.Because this study and others [l-4] show a dis-tinction between mammalian cells, especially nor-mal and tumour cells, this phenomenon deservesfurther evaluation.

References

W. Scholz, U. Staskiewicz, F. A. Popp and W. Nagl, Light-stimulated ultraweak photon re-emission of human amnioncells and Wish cells, Cell Biophys., 13 (1988) 55-63.R. van Wijk and D. H. J. Schamhart, Regulatory aspects oflow intensity photon emission, Experientia, 44 (1988) 586-593.R. van Wijk and H. van Aken, Light-induced photon emissionby rat hepatocytes and hepatoma cells, Cell Biophys., 18 (1991)15-29.H. J. Niggli, Biophoton re-emission studies in carcinogenicmouse melanoma cells, in F. A. Popp, Q. Gu and K. H. Li(eds.), Recent Advances in Bi ophoton Research and its Appli -cation, World Scientific, Singapore, London, 1992, pp. 231-242.F. A. Popp, B. Ruth, W. Bahr, J. Bohm, P. Grass, P. Grolig,M. Rattemeyer, H. G. Schmidt and P. Wulle, Emission ofvisible and ultraviolet radiation by active biological systems,Collect. Phenomena, 3 (1981) 187-214.H. Inaba, Super-high sensitivity systems for detection andspectral analyses of ultra weak photon emission from biologicalcells and tissues, Experi entia, 44 (1988) 55G559.

I

8

9

10

11

12

13

14

15

D. H. J. Schamhart, W. Berendsen, J. van Rijn and R. vanWijk, Comparative studies of heat sensitivity of several ratphenomena cell lines and hepatocytes in primary culture,Cancer Res., 44 (1984) 4507-4516.J. E. M. Souren, C. Schneijdenberg, A. Verkleij and R. vanWijk, Factors controlling the rhythmic contraction of collagengels by neonatal heart cells, In vitro Cell. Dev. Biol., 2&A(1992) 199-204.I. Harary and B. Farley, In vitro studies on single beatingrat heart cells. I. Growth and organization, &D. Cell Res.,29 (1963) 451465.J. E. M. Souren, M. Ponec and R. van Wijk, Contraction ofcollagen by human fibroblasts and keratinocytes, In vitro Cell.Dev. Biol., 25 (1992) 1039-1045.R. Hay, M. Macy, T. R. Chen, P. McClintock and Y. Reid,American Type Culture Collection, Catalogue of Cell Linesand Hybridomas, ATCC, 12301 Parklawn Drive, Rockville,MD, 6th edn., 1988.H. C. Pitot, C. Peraino, P. A. Morse and V. R. Potter,Hepatomas in tissue culture compared with adapting liver invivo, Natl. Cancer I nst. Monogr., 13 (1964) 229-242.E. B. Thompson, G. M. Tomkins and J. F. Curran, Inductionof tyrosine a-ketoglutarate transaminase by steroid hormonesin a newly established tissue culture cell line, From. Natl.Acad. Sci. USA, 56 (1966) 296303.H. G. Keizer, G. J. Schuurhuis, H. J. Broxtennans, J. Lankelma,W. G. E. J. Schoonen, J. van Rijn, H. M. Pinedo and H.Joenje, Correlation of multidrug resistance with decreaseddrug accumulation, altered subcellular drug distribution, andincreased P-glycoprotein expression in cultured SW-1573 hu-man lung tumor cells, Cancer Res., 49 (1989) 2988-2993.F. Grasso, C. Grillo, F. Musumeci, A. Triglia, G. Rodolico,F. Cammisuli, C. Rinzivillo, G. Fragati, A. Santuccio and M.Rodolico, Photon emission from normal and tumor humantissues, Experientia, 48 (1992) l& 13.

VanWijk et al (1993)-Light-induced photon emission by mammalian cells.pdf

Documents