8
Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase Gautam Banerjee 1 , Nishma Gupta 1,2 , Jyoti Tiwari 3 , Govindarajan Raman 1 1 Cell and Molecular Biology, Toxicology Section, Environmental Safety Laboratory, Hindustan Lever Research Centre, Andheri, Mumbai, India, 2 Department of Biotechnology, Madurai Kamraj University, Madurai, India, and 3 Tea Science Laboratory, Unilever Research, Bangalore, India The normal human keratinocyte cell line, HaCaT, was transformed using multiple doses of ultraviolet (UV)A1B (UVA, 150–200 mJ/cm 2 and UVB, 15– 20mJ/cm 2 6). Malignant transformation was con- firmed by upregulation of Cyclin D1 (mRNA) and formation of colonies on soft agar. To identify the genes involved in this transformation process, we have done rapid amplification of polymorphic DNA using RNA from unexposed and multiple-exposed cells. Six percent PAGE showed several differentially regulated genes in exposed cells compared with unexposed cells. Total 19 genes were identified, cloned and sequenced. Three of these 19 cloned genes showed 99% homology at both DNA and protein levels to a stretch of 540 bp (180 aa) of long interspersed element (LINE)-1 reverse transcrip- tase (RT) open reading frame (ORF-2). Colonies from soft agar showed upregulation of this gene compared with non-colonized (lawn on soft agar) cells as detected by RT-PCR. This data implicates LINE-1 RT (ORF-2) in UV-induced malignancy and can possibly be used as a marker for the diagnosis of UV-induced skin cancer. Key words: HaCaT; keratinocytes; LINE-1 reverse transcriptase; RAPD; skin cancer; soft agar; UV. C ell death or ‘Apoptosis’ is an essential physiolo- gical process that occurs in metazoans for their normal growth and development, regulation of cell turnover and as a defense strategy against invading pathogens. Deregulation of apoptosis leads to disrup- tion of tissue homeostasis giving rise to a variety of disorders including cancer, neuro-degenerative dis- eases, autoimmune diseases and diabetes (1). Mal- function of apoptosis leading to cancer could involve stepwise mutations in proto-oncogenes (ras, c-myc) (2), tumor-suppressor genes (p53) (3), anti-apoptotic genes (Bcl-X L ) (4) or overexpression of genes (telo- merase, cytokeratins) (5–7). The defective regulation of apoptosis may contribute to the etiology of cancer, and the impairment of normal cell death processes has been implicated in neo-plastic transformation as several studies have shown (8, 9). Ultraviolet (UV) radiation in particular UVB (290– 320 nm) from sunlight represents one of the most important external stimuli that affects skin by inducing immuno-suppression, cancer, premature skin aging, inflammation and cell death (10). UV radiation from sun has also been well established as a principal carcinogen serving as initiator and promoter for many skin tumors (11). It can modulate both life/death signaling processes in either in vivo or in vitro systems. Studies have shown that UVB irradiation causes apoptosis, necrosis, differentiation and malignancy in keratinocytes in a dose-dependent manner (12, 13). HaCaT cells constitute the basis of the in vitro transformation assay for detecting carcinogens. The end point used in this assay is the morphological transformation of colonies that develop 21 days after the seeding of HaCaT cells postexposure. The relevance of the transformed phenotype as an indicator of neo-plastic properties has been demon- strated by their ability to produce tumor when injected into isolog animals (14, 15). The study aims to identify novel genes that are regulated during this process so that they can be used (1) as markers or (2) to identify new pathways. We have shown that with repeated subapoptotic doses of UV some cells become transformed cells and survive as colonies on soft agar, while at higher doses cells die Abbreviations: ORF, open reading frame; RAPD, rapid amplification of polymorphic DNA; RT, reverse transcriptase. Photodermatol Photoimmunol Photomed 2005; 21: 32–39 Blackwell Munksgaard Copyright r Blackwell Munksgaard 2005 32

Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

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Page 1: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

Ultraviolet-induced transformation of keratinocytes: possible involvement of

long interspersed element-1 reverse transcriptase

Gautam Banerjee1, Nishma Gupta1,2, Jyoti Tiwari3, Govindarajan Raman1

1Cell and Molecular Biology, Toxicology Section, Environmental Safety Laboratory, Hindustan Lever Research Centre, Andheri, Mumbai, India,2Department of Biotechnology, Madurai Kamraj University, Madurai, India, and 3Tea Science Laboratory, Unilever Research, Bangalore, India

The normal human keratinocyte cell line, HaCaT, was

transformed using multiple doses of ultraviolet

(UV)A1B (UVA, 150–200mJ/cm2 and UVB, 15–

20mJ/cm2 � 6). Malignant transformation was con-

firmed by upregulation of Cyclin D1 (mRNA) and

formation of colonies on soft agar. To identify the genes

involved in this transformation process, we have done

rapid amplification of polymorphic DNA using RNA

from unexposed and multiple-exposed cells. Six percent

PAGE showed several differentially regulated genes in

exposed cells compared with unexposed cells. Total 19

genes were identified, cloned and sequenced. Three of

these 19 cloned genes showed 99% homology at both

DNA and protein levels to a stretch of 540 bp (180 aa) of

long interspersed element (LINE)-1 reverse transcrip-

tase (RT) open reading frame (ORF-2). Colonies from

soft agar showed upregulation of this gene compared

with non-colonized (lawn on soft agar) cells as detected

by RT-PCR. This data implicates LINE-1 RT (ORF-2)

in UV-induced malignancy and can possibly be used as a

marker for the diagnosis of UV-induced skin cancer.

Key words: HaCaT; keratinocytes; LINE-1 reverse

transcriptase; RAPD; skin cancer; soft agar; UV.

Cell death or ‘Apoptosis’ is an essential physiolo-

gical process that occurs in metazoans for their

normal growth and development, regulation of cell

turnover and as a defense strategy against invading

pathogens. Deregulation of apoptosis leads to disrup-

tion of tissue homeostasis giving rise to a variety of

disorders including cancer, neuro-degenerative dis-

eases, autoimmune diseases and diabetes (1). Mal-

function of apoptosis leading to cancer could involve

stepwise mutations in proto-oncogenes (ras, c-myc)

(2), tumor-suppressor genes (p53) (3), anti-apoptotic

genes (Bcl-XL) (4) or overexpression of genes (telo-

merase, cytokeratins) (5–7). The defective regulation

of apoptosis may contribute to the etiology of cancer,

and the impairment of normal cell death processes has

been implicated in neo-plastic transformation as

several studies have shown (8, 9).

Ultraviolet (UV) radiation in particular UVB (290–

320 nm) from sunlight represents one of the most

important external stimuli that affects skin by

inducing immuno-suppression, cancer, premature skin

aging, inflammation and cell death (10). UV radiation

from sun has also been well established as a principal

carcinogen serving as initiator and promoter for many

skin tumors (11). It can modulate both life/death

signaling processes in either in vivo or in vitro systems.

Studies have shown that UVB irradiation causes

apoptosis, necrosis, differentiation and malignancy in

keratinocytes in a dose-dependent manner (12, 13).

HaCaT cells constitute the basis of the in vitro

transformation assay for detecting carcinogens. The

end point used in this assay is the morphological

transformation of colonies that develop 21 days after

the seeding of HaCaT cells postexposure. The

relevance of the transformed phenotype as an

indicator of neo-plastic properties has been demon-

strated by their ability to produce tumor when

injected into isolog animals (14, 15).

The study aims to identify novel genes that are

regulated during this process so that they can be used

(1) as markers or (2) to identify new pathways. We

have shown that with repeated subapoptotic doses of

UV some cells become transformed cells and survive

as colonies on soft agar, while at higher doses cells dieAbbreviations: ORF, open reading frame; RAPD, rapidamplification of polymorphic DNA; RT, reverse transcriptase.

Photodermatol Photoimmunol Photomed 2005; 21: 32–39Blackwell Munksgaard

CopyrightrBlackwellMunksgaard 2005

32

Page 2: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

by either apoptotic or necrotic pathways. In the

present report we present data on transformation of

normal human keratinocytes, HaCaT cell line by

multiple subapoptotic doses of UV. We have also

identified and characterized a gene, which is differen-

tially regulated during this transformation process.

Materials and methodsMaterials

DMEM medium, RPMI-1640 medium, antibiotic–

antimycotic solution and TRI reagent were purchased

from Sigma, St Louis, MO, USA. Fetal calf serum was

from Hy-clone, Logan, UT, USA. Reverse transcriptase

(RT) kit, Taq DNA polymerase, was purchased from

MBI Fermentas, Hanover, MD, USA. Gel extraction

and plasmids isolation kit were purchased from Qiagen,

Valencia, CA, USA. pGEM-T easy vector was pur-

chased from Promega, Madison, WI, USA. Custom-

made synthesis of primers and sequencing of DNA was

done by Microsynth, Balgach, Switzerland. LB agar

and yeast extract were from Hi-Media, Mumbai, India.

UV exposure was done using Woton lamp at a

distance of 26 cm and the dose was monitored using

UV meter (Solar Light Inc., Philadelphia, PA, USA).

HaCaT cell line was a gift from Dr. N. Fusenig,

Germany.

Methods

Cell culture: HaCaT and A431 were maintained in

complete DMEM in the presence of 10% FCS at

37 1C and 5% CO2. They were subcultured in every

3–4 days using Trypsin EDTA.

UV exposure to HaCaT cells: Confluent layers of

HaCaT cells grown in a six-well plate were exposed to

UVA1B (UVB, 15–20mJ/cm2). These cells were

cultured in fresh medium for 1–2 days and re-exposed

using the same dose. This procedure was repeated six

times. After each exposure, cells were trypsinized and

divided into three aliquots. These aliquots were used

for RNA extraction, soft agar colony assay and the

remaining aliquot was used for the next exposure.

Control cells were subjected to similar procedures

without being UV exposed.

Soft agar colony-forming assay: Soft agar colony-

forming assay using multiple-exposed cells was done

as follows. Base agar plates were prepared containing

2.5ml of 1% agar and 2.5ml of 1 � RPMI with 15%

FCS. HaCaT cells (1 � 106 cells/90mm plate) were

suspended in 1ml of RPMI with 15% FCS and 1ml

of 0.7% agar and were plated. These plates were kept

at 37 1C for 21 days and examined for colonies.

Individual colonies as well as the lawn (non-colo-

nized) cells were picked up and RNA was isolated

from these cells for further analysis.

PCR amplification: RNA was extracted from the

exposed as well as unexposed cells using TRI reagent

as per manufacturer instruction. 1.5 mg RNA was

converted to cDNA using the RT kit from MBI

Fermentas. PCR was done using the b-actin primer to

equalize the amount of cDNA for subsequent

analysis. PCR was also performed without converting

them to cDNA to determine the effect/contamination

of genomic DNA in the extracted RNA.

PCR was conducted in 10 � PCR buffer with

25mM MgCl2 supplemented with 2mM dNTP’s,

20 pmol sense and antisense primers for b-actin or

Cyclin D1, 1 ml cDNA containing the same amount

and 1 unit of Taq DNA polymerase in a 20 ml reactionmixture. PCR profile consisted of 25 cycles (for b-actin) and 35 cycles (for other genes) of 94 1C for 60 s,

57 1C for 90 s, 72 1C for 2min and final extension at

72 1C for 10min. We have checked that the condition

we have used for (for b-actin) lies in the exponential

phase and not in the stationary phase.

PCR products were analyzed on a 2% agarose gel

and stained with ethidium bromide and visualized

under UV illumination.

The sequences of the primers are given below:

b-actin

Forward 50-AGCGGGAAATCGTGCGTG

Reverse 50-CAGGGTACATGGTGGTGCC

Cyclin D1

Forward 50-AGGAGAACAAACAGATCA-30

Reverse 50-TAGGACAGGAAGTTGTTG-3 0

Rapid amplification of polymorphic DNA (RAPD):

The cDNA that were free from contamination DNA

as judged by – RT-PCR experiments were used for all

RAPD – PCR analysis. This confirms the absence of

any artifact DNA or genomic long interspersed

element (LINE)-1 sequences. The cDNA from un-

exposed and exposed cells was diluted to 1 : 50, 1 : 75

and 1 : 100. cDNA was amplified using KD1 (50-

AAGAGCCCGT-30) primer for 35 cycles as described

below – 95 1C for 5min, 36 1C for 30 s and 72 1C for

30 s followed by 35 cycles of 95 1C for 1min, 36 1C for

30 s and 72 1C for 30 s. The amplified products were

separated on 6% SDS-PAGE and silver stain was

done. The differentially expressed bands were cut out,

extracted and purified. The purified DNA was

33

Ultraviolet-induced transformation of keratinocytes

Page 3: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

re-amplified using the same procedure. Two percent

agarose gel was used to separate the DNA and

amplified band was excised from the gel and was

extracted using gel extraction kit (Qiagen). The DNA

was ligated in pGEM T easy vector (Promega)

overnight at 4 1C. DH5a was used as host for

transformation of ligated plasmids.

Isolation of plasmids: Plasmids were isolated from

overnight culture of transformed DH5a using the spin

mini prep kit (Qiagen). The plasmids were digested

with EcoR1 to confirm the presence of insert. Once

confirmed the inserts were sent for sequencing.

Data analysis: The sequence was analyzed using the

National Center for Biotechnology (NCBI) database

and the alignments were done using Clustlaw data-

base of the European Biochemical Institutes.

LINE-1 regulation in multiple-exposed cells: LINE-1

RT sequence specific primers were designed (forward

50-ACGGTGATTTCTGCATTTCC-3 0 and reverse

50-TTTCTTAAGCCGGTCTGAAA-30) from pub-

lished sequences and PCR was done using cDNA

from exposed cells (after the fifth exposure), un-

exposed cells as well as the cells from lawn and

colonies obtained from soft agar plates.

ResultsThe generation of malignant colonies on soft agar is a

useful method to identify malignant clones. The

transformation of normal HaCaT to malignant

phenotype by UVB was investigated by soft agar

colony assay. Unexposed cells did not show any

colony even after 6 weeks. The colonies start

appearing from the fourth exposure onward. The

numbers (not detected at unexposed cells to about

2000/90mm plate after the fifth exposures) as well as

the size of the colonies increases with increasing the

number of exposure. The unexposed cells were taken

from the same passage as that of cells after the fifth

exposure, which rules out the involvement of passage

number in colony formation. A431 cells, known

carcinoma cells, were used as positive control (with-

out any UV exposure). The result is shown in Fig. 1.

All the pictures were taken in the same magnification

(� 40). A431 is a cancer cell line hence the increased

number of colony is justified, whereas small percen-

tages of HaCaT (4–5%) are converted to malignant

phenotype. Hence the numbers of colonies are less in

HaCaT and should not be compared with that of

A431cells.

The upregulation of Cyclin D1 was seen in cells

obtained from liquid culture medium (after the fifth

exposure compared with unexposed cells). The results

a b c

d e f

Fig. 1. Soft agar colony assay using ultraviolet (UV)-exposed and unexposed HaCaT cells. (a) Unexposed cells, (b)the first exposure, (c) the second exposure, (d) the fourth exposure, (e) the fifth exposure, (f) A431 cells. Confluentlayers of HaCaT cells grown in a six-well plate were exposed to UVB (15–20mJ/cm2). These cells were cultured infresh medium for 1–2 days and re-exposed using the same dose. This procedure was repeated six times for a periodof 2 weeks. Control cells were subjected to similar procedures without being UV exposed. The colonies wereallowed to grow for 21 days post last exposure. The pictures were taken at the same magnification (� 40).

34

Banerjee et al.

Page 4: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

are shown in Fig. 2. Upregulation was observed in the

multiple-exposed cells compared with unexposed cells

from the same passage.

To identify the regulation of genes in this transfor-

mation process we have done RAPD using different

dilutions of cDNA from the exposed (the first to fifth

exposure) and unexposed cells. Differential expression

of bands of varying sizes was seen. The bands that

were either new or upregulated (500–750 bp) were

taken out for further analysis. Total 19 bands were

identified. Bands from different exposures (fourth

and fifth) with approximate 550 bp were re-amplified

and purified from a 2% agarose gel. The DNA was

ligated in pGEM -T easy vector. The presence of insert

was confirmed by digesting the plasmids with EcoR1.

The insert was sequenced (Microsynth). Three

different sets of inserts (isolated from cells post fourth

and fifth exposures) had significant homology (95%)

among themselves in both DNA as well as in protein

sequence. The protein sequence homology of the

cloned genes is shown in Fig. 3. The description of the

LINE-1 open reading frame (ORF-2) gene is shown in

Fig. 4. Blast analysis using the NCBI database

showed homology (96%) with the OFR-2 of the

LINE-1 RT (Fig. 5). Hence three out of the 19 cloned

gene were similar to LINE-1, ORF-2.

In order to identify the presence of ORF-2 of RT in

both exposed/un-exposed cells as well as in the cells

from the soft agar colonies (single colony and lawn),

a sequence-specific primers pair was designed. The

primer pair was designed in such a way that the

forward primer situated upstream of our cloned

gene and the reverse primer were inside the cloned

gene. Hence any upregulation indicates the regulation

of entire gene and not a spliced part. The ORF-2 was

upregulated in exposed cells compared with unex-

posed cells. The same assay was done using the

colonies and lawn cells. The ORF-2 RT was also

upregulated in the colonies compared with the lawn or

non-colony cells. The data is shown in Fig. 6.

DiscussionUV radiation from sun is one of the major environ-

mental agents that affects skin. Apart from its critical

role in formation of vitamin D, it adversely affects

mammalian systems by causing sunburn, suntanning,

immunosuppression and most importantly it is

implicated in induction of non-melanoma skin cancer.

Unexposed

Cyclin D1

β-actin

Exposed

Fig. 2. Upregulation of Cyclin D1. (a) Cyclin D1 inunexposed and exposed cells, (b) b-actin in unexposedand exposed cells after the fifth exposure. Confluentlayers of HaCaT cells grown in a six-well plate wereexposed to UVB (15–20mJ/cm2). These cells werecultured in fresh medium for 1–2 days and re-exposedusing the same dose. This procedure was repeated sixtimes for a period of 2 weeks. Control cells were sub-jected to similar procedures without being UV exposed.

g16 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNRTEPSEIRPHIYNHL 60g17 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNZTEPSEIRPHIYNHL 60g5 IAKTALSKKNKAGGIMLPDFKLHYKDTVTKTAGYCYQNRHIDQZNRTEPSEIRPHIYNHL 60 ********************************************* **************

g16 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120g17 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120g5 IFDKPDKNKKWGNDSLFNKZCWENWLAICRKLKLDPFLTPYTKINSRWIKDIHVRPETIK 120 ************************************************************

g16 TLKENLGNSIQDIGMGKDSMTKTAKAMATKAKIDKWDLIKLKSLCTAKKK---KKNKKNK 177g17 TLKENLGNSIQDIGMGKDSMTKTAKAMATKAKIDKWDLIKLKSLCTAKKK---QKKQKNK 177g5 TLKENLGNSIQDIGMGKDSMTKTAKSNGNKSQNZZMGSNZTKEPLPSKKKKKKQKNQKNK 180 *************************: ..*:: . *. .:***...:*::***

g16 KZTKNYHQS 186 g17 QKTIIRVK- 185 g5 QKTIIRV-- 187 : *

Fig. 3. Protein sequence alignment of cloned genes (g-5, 16 and 17). The sequence homology was done using theclustlaw software.

35

Ultraviolet-induced transformation of keratinocytes

Page 5: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

An effective repair system that excises the photo-

products formed primarily at sites of adjacent

pyrimidines of DNA operates in cells (3). If UV-

exposed cells fail to repair the damaged DNA, then

p53-dependent apoptosis is induced in keratinocytes,

which serves as a protective mechanism against

UTR ORF1 ORF2 UTRA(n)

3’5’

pL1

1 239 380 480 498 590 773 1130 1147 1275 aa

EN Z RT CYS - rich

RT5Z8

a

b

Fig. 4. Gene sequence of long interspersed element-1 reverse transcriptase (RT). (a) Full-length gene having openreading frame (ORF)-1 and ORF-2 with non-coding region at both 50 and 30 region and a promoter region (Pl-1),(b) full length of ORF-2 �239 amino acid long endonuclease domain (EN), octapeptide repeat region (Z), RTregion and a cysteine-rich residue. The crossed bar on top of RT region indicates the cloned gene.

ORF-2 AAGTTCATATGGAACCAAAAAAGAGCCCGCATTGCCAAGTCAATCCTAAGCCAAAAGAAC 1800 g17 ------------------------------ATTGCCAAGACAGCCCTAAGTAAAAAGAAC 30

********* ** ****** ********ORF-2 AAAGCTGGAGGCATCACACTACCTGACTTCAAACTATACTACAAGGCTACAGTAACCAAA 1860 g17 AAAGCTGGAGGCATCATGCTACCTGACTTCAAACTACATTACAAGGATACAGTAACAAAA 90

**************** ****************** * ******* ********* ***ORF-2 ACAGCATGGTACTGGTACCAAAACAGAGATATAGATCAATGGAACAGAACAGAGCCCTCA 1920 g17 ACAGCAGGGTACTGTTACCAAAACAGACATATAGACCAGTAGAACTGAACAGAGCCCTCA 150

****** ******* ************ ******* ** * **** **************ORF-2 GAAATAATGCCGCATATCTACAACTATCTGATCTTTGACAAACCTGAGAAAAACAAGCAA 1980g17 GAAATAAGGCCCCATATCTACAACCATCTGATCTTTGACAAACCTGACAAAAACAAGAAA 210

******* *** ************ ********************** ********* **ORF-2 TGGGGAAAGGATTCCCTATTTAATAAATGGTGCTGGGAAAACTGGCTAGCCATATGTAGA 2040 g17 TGGGGAAACGATTCCCTATTTAATAAATGATGCTGGGAAAACTGGCTAGCCATATGTAGA 270

******** ******************** ******************************ORF-2 AAGCTGAAACTGGATCCCTTCCTTACACCTTATACAAAAATCAATTCAAGATGGATTAAA 2100g17 AAGCTGAAACTGGATCCCTTCCTTACACCTTATACAAAAATTAATTCAAGATGGATTAAA 330

***************************************** ******************ORF-2 GATTTAAACGTTAAACCTAAAACCATAAAAACCCTAGAAGAAAACCTAGGCATTACCATT 2160 g17 GACATACACGTTAGACCTGAAACCATAAAAACCCTAAAAGAGAATCTAGGCAATTCCATT 390

** ** ****** **** ***************** **** ** ******* * *****ORF-2 CAGGACATAGGCGTGGGCAAGGACTTCATGTCCAAAACACCAAAAGCAATGGCAACAAAA 2220g17 CAGGACATAGGCATGGGCAAAGACTCCATGACTAAAACAGCAAAAGCAATGGCAACAAAA 450

************ ******* **** **** * ****** ********************ORF-2 GACAAAATTGACAAATGGGATCTAATTAAACTAAAGAGCTTCTGCACAGCAAAAGAAACT 2280 g17 GCCAAAATTGATAAATGGGATCTAATTAAACTAAAGAGCCTCTGCACAGCAAAAAAAA-- 508 *

********* *************************** ************** ***ORF-2 ACCATCAGAGTGAACAGGCAACCTACAACATGGGAGAAAATTTTCGCAACCTACTCATCT 2340g17 --- AACAAAAAAAACAAAAAA---ATAA-ACAAAAAACTATCATCAGAGTGAA------- 554

* ** * **** ** * ** * * * ** ** * *

Fig. 5. Sequence alignment of open reading frame (ORF)-2 of long interspersed element-1 reverse transcriptase andcloned gene (g-17).

36

Banerjee et al.

Page 6: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

development of cancer (3). UV-induced DNA damage

can lead to mutations that may trigger malignancy.

UV-induced skin cancers in mice models have earlier

been analyzed. It is reported that both ras and p53

genes were mutated in these models (16). As a result

of such DNA modification, the apoptosis pathway is

inhibited and malignancy allowed developing.

Mutations in tumor-suppressor gene, p53, have

been found to be characteristic in UV-induced skin

cancer. Evidences also have shown that p53 mutations

offer selective growth advantage to initiated cells.

Cells even with a single p53 mutation have less

apoptotic cell death in comparison with neighboring

p53�/� cells, thereby they continue to survive and

multiply (17). This would mimic an in vivo condition

where UV light would exert selective pressure for

mutated (p53�/�) keratinocytes hence allowing the

cells to clonaly expand and eventually become

malignant. These cells still may undergo apoptosis

by p53-independent pathways.

In this paper we have shown that repeated exposure

at subapoptotic doses of UV (UVA, 150–200mJ/cm2

and UVB, 15–20mJ/cm2) might cause transformation

in HaCaT. It is reported that HaCaT is p53�/� but it

cannot spontaneously transform during multiple

passages (18). Tumorigenic transformation in HaCaT

has been induced by ras oncogene transfection and

at elevated temperatures (18). The transformed cells

we have generated because of multiple exposures are

not because of spontaneous transformation through

multiple passages as it is reported that up to at least

300 passages this type of transformation does not take

place (18).

Telomerase is an enzyme known to protect

immortal/malignant cells from loss of telomeric

repeats at the end of chromosomes and thereby

protects the cells from death. All malignant cells/

tissues are known to contain high levels of telomerase

while the normal cells/tissues are lacking in this

enzyme (5, 6). It is reported that unexposed HaCaT

possess some basal level of telomerase activity while

those of malignant cell lines like A431 or HeLa

possess much higher amount (19). This data is

consistent with our findings on soft agar colony-

forming assay. We have shown that unexposed cells

did not induce colony formation while multiple-

exposed cells induced colony formation on soft agar

plates. With increasing number of exposures (beyond

the fourth exposure), the number and size of the

colonies increase. These data demonstrate the trans-

formation of HaCaT from non-malignant to a

malignant phenotype by multiple UVB exposure.

The neo-plastic transformation of human epidermal

keratinocytes by ionizing radiation has also been

reported and soft agar colony assay was done to

confirm the transformation by radiation (20).

LINES are most abundant mobile elements in

mammalian genome. They are non-viral retrotran-

sposons occurring as moderately repeated sequences

in the genome. Full-length version of this gene is

approximately 7 kb and include a 5 0 non-coding

region with a promoter, ORF-1 and ORF-2 and a 3 0

non-coding region ending with an A-rich stretch.

Most common LINE constitute the L1 LINE

family, the consensus sequence of which contains

ORF-2 coding for RT. RT plays an important

role in the replication of these retro elements by

reverse transcribing their template RNA into double-

strandeded DNA that is ultimately integrated into

the host genome by the element encoded integrase

(21, 22).

Human LINE-1 retrotransposons have known

implications in origin and progression of tumors by

either causing insertional mutations or chromosomal

translocations/rearrangements (23, 24). These ele-

ments have been shown to be expressed in a variety

of adult and pediatric germ cell cancers (24, 25).

Expression of these retrotransposons has been shown

to be favored in cells of germ LINE origin and also in

tumors of epithelial origin (22). LINE-1 transposition

is also shown to cause chromosomal rearrangement

giving rise to fusion transcript encoding an aberrant

transcription factor, which is implicated in desmo-

plastic round cell tumor (23, 26). RT is thus known

to have significant biological role in the etiology

of tumors.

Distinct upregulation of LINE-1 is observed in the

set of multiple-UV-exposed cells as compared with

unexposed cells. Treatment of cells with UV is known

Unexposed ExposedLane 1 2 1 2 3 4

a b

c d

Unexposed Exposed

LINE 1, ORF 2

Lawn colony colony colony

Lawn colony colony colony

β-actin

Fig. 6. Upregulation of long interspersed element(LINE)-1 reverse transcriptase open reading frame(ORF)-2 in the multiple-UV-exposed cells. (a,c) Lane-1: unexposed cells, lane-2: exposed cells. (b,d) Lane-1:non-colonized cells (exposed) from soft agar plate,lanes 2–4: different colonies from soft agar plate(exposed). (a,b) ORF-2; (c,d) b-actin.

37

Ultraviolet-induced transformation of keratinocytes

Page 7: Ultraviolet-induced transformation of keratinocytes: possible involvement of long interspersed element-1 reverse transcriptase

to enhance LINE-1 activity in embryonal carcinoma

cells (27). We have shown that, of the multiple

exposed populations, only the malignant population,

which formed colonies on soft agar, showed increase

in LINE-1 gene expression as compared with the non-

malignant population. Thus UV enhances LINE-1

activity, which in turn may cause alterations and

modifications in the genome eventually leading to

malignancy. Expression of LINE-1 and ORF-2 thus

can be used as a potential marker to probe UV-

induced malignancy. Its implication in chemically

induced malignancy is under investigation.

We have recently cloned one gene from the

magnetically purified non-apoptotic population (UV

was used to induce apoptosis) from the UV-exposed

apoptotic cells (28). This population being non-

apoptotic, it has a tendency of transformation and

showed upregulation of LINE expression (data not

shown). It is possible that this population (UV

exposed but resistant to apoptosis) may be trans-

formed toward malignancy once exposed to multiple

UV doses. Recently, It has been shown that ZnCl2 can

induce syrian hamster embryo cell transformation

(29). Although ZnCl2 is known for its anti-apoptotic

effect, the cell that survived has a high possibility of

transformation toward malignant phenotype. This

data is consistent with our findings.

It can be summarized that UVB can induce

transformation of human keratinocytes involving

LINE-1. Those cells, which can escape apoptotic

death by UV, are more resistant to UV-induced death

and possibly are more prone to malignant transfor-

mation using LINE-1 RT.

The presence/involvement of other genes in the

elicitation of malignancy as well as possibility of using

them as a predictive tool for carcinogenesis is under

investigation.

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Accepted for publication 15 September 2004

Corresponding author:

Gautam Banerjee

Cell and Molecular Biology

Toxicology Section

Environmental Safety Laboratory

Hindustan Lever Research Centre

B.D. Sawant Marg, Andheri

Mumbai

India

Tel: 191 22 2827 6370

Fax: 191 22 2836 3680

e-mail: [email protected]

39

Ultraviolet-induced transformation of keratinocytes