Cell Culture

All cells were grown in a humidified incubator at 37®C in 5 %C0 2- Media and

solutions used are listed in Appendix 1.1.

2.1.1 Routine Cell Culture

The cells that were used were human kératinocytes. Normal kératinocytes were from neonatal foreskins and adult buccal mucosa and the origins of the squamous cell carcinoma cell lines are listed in Table 2.1.

2.1.1.1 Normal Keratinocyte Subculture

Normal kératinocytes from human foreskins and normal oral kératinocytes from buccal mucosa were cultured and passaged as described by Watt (1994). (Briefly, primary cultures were produced by chopping epidermis into fine pieces and treating them with trypsin to produce a suspension of kératinocytes, which were then plated onto feeder cells prepared as described in Section 2.1.1.2). Subconfluent or just confluent cultures were washed in 5ml of versene and then incubated with 5ml of versene for 5 minutes to remove any remaining feeder cells. The versene with the feeder cells was then discarded and a 1 + 4 trypsin + versene mixture added to the kératinocytes for about 5-10 minutes at 37°C. Once the cells had lost their attachment to the flask they were transferred to centrifuge tube with 10ml of FAD -I- PCS -I- HICE

and spun at l(XX)rpm for 5 minutes. After discarding the supernatant the cells were

resuspended in FAD -I- PCS -f- HICE and then seeded onto a mitomycin C-treated J2 feeder layer (see below) at 2 x 10^ cells per 75cm^ flask, 5 x 10^ - 1 x 10^ cells per

25cm^ flask. Cultures were fed three times a week with fresh FAD + PCS 4- HICE.

Materials and Methods

Small colonies generally appeared within 3-4 days and the flasks were confluent in 8-10 days.

The foreskin keratinocyte strains kb, kk and kq were used in these experiments

up to passage 6 and oral keratinocyte strains 6062 and 796 were used up to passage 6 .

2.1.1.2 Preparation of 3T3 Feeder Cells

Clone J2 of 3T3 Swiss mouse embryo fibroblasts are a clone selected for their ability to support keratinocyte growth. J2s were routinely cultured to act as stocks of feeder layers for keratinocyte culture (Watt 1994). J2s were cultured in E4 4- 10% DCS and subcultured at 1:10 every 5-6 days. Confluent J2s were mitotically inactivated by adding 4/xg/ml mitomycin C to the culture medium and incubating at 37®C for 2 hours. The cells were washed in versene, trypsinised, spun down at lOOOrpm for 5 minutes, resuspended in FAD + FCS 4- HICE and then seeded at 1:3 in 25cm^ tissue culture flasks. Cells were then incubated at 37°C for 2-24 hours prior to seeding kératinocytes.

2.1.1.3 Culture of Malignant Keratinocyte Cell Lines

Oral squamous cell carcinoma (SCC) cell lines H357 and H376 (Prime et al.

1990) were the kind gift of Professor S.S. Prime. These cell lines are feeder

independent and were cultured in FAD 4- FCS 4- HICE medium. The method of subculture was the same as for normal kératinocytes but it was not necessary to remove feeder cells before trypsinisation, and the malignant kératinocytes were plated directly onto the tissue culture flasks at the same density used for normal kératinocytes.

Materials and Methods

Table 2.1 Characteristics of Malignant Cell Lines (Data from Prime et al. 1990)

Cell Line Site' Pathology ^ Tumorigenic in Nude Mice

H357 Tongue WD /I Yes

H376 FOM WD/III No

^FOM: Floor of mouth ^WD: Well differentiated

Roman Numerals refer to tumour stage:

I:Tumour <2cm in its greatest dimension, no regional lymph node involvement, no métastasés.

Ill [Tumour <4cm, with regional lymph node involvement, no distant métastasés.

2.1.1.4 Freezing Down Cell Stocks

After trypsinisation and washing in complete medium cells were resuspended at 10^ per ml in 1:10 DMSO:FCS in a Nunc cryotube in an insulated container and placed at -70°C for 24 hours. After this time the vials were transferred to liquid nitrogen for storage.

2.1.1.5 Thawing Cell Stocks

Vials of frozen stocks were thawed individually at 37®C to avoid the risk of cross-contaminating cell types and strains. As soon as the vial contents were thawed they were gently resuspended in complete medium (E4 + 10% DCS for J2s and standard FAD 4- FCS 4- HICE for kératinocytes) and spun down at KXXlrpm for 5 minutes. The supernatant was aspirated off and the remaining cells were resuspended

Materials and Methods

in fresh medium, counted and plated out.

2.1.2 Transfection of Integrin Subunits into the Cell Lines

H357 is deficient in the «y integrin subunit and H376 has a low level of

expression of the B4 integrin subunit (Sugiyama et al. 1993). As such these cell lines

provide a model to study the effect of integrin subunits on cell behaviour.

The H357 cell line was transfected with human integrin «y cDNA (Loftus et al.

1990) and the H376 cell line with human integrin cDNA (Giancotti et al. 1992).

A full length «y cDNA, kindly provided by J. Loftus, Scripps Research Institute, was subcloned in to pRc/CMV (Invitrogen Corp.) by John Marshall (ICRF). «y was expressed under the control of the CMV promoter; the plasmid also contained a

neomycin resistance-gene as a selectable marker. The B4 cDNA was kindly provided

by F. Gianocotti, Kaplan Cancer Center. 20^g plasmid DNA, purified using caesium chloride, was transfected into 2.5 x 10^ cells in a 100mm dish using cadcium phosphate

by Masaru Sugiyama (ICRF) as described below. Cells were seeded into 100mm

dishes, fed with FAD + FCS + HICE and left to become confluent for 1 day. 20pg of DNA was used per transfection; the volume of DNA was made up to 160/xl with 1/10 TE pH 7.6 (Appendix 1.2), mixed well and incubated at 38°C for 5 minutes. 160/xl of pre-warmed CaClz (Appendix 1.2) was added to the DNA, mixed well and then incubated for 10 minutes at 38°C. 320^1 of pre-warmed 2X HBS (Appendix 1.2)

was added drop-wise while gently vortexing the mixture. The mixture was then

vortexed to ensure good mixing, incubated in a 38°C water bath for 15 minutes, and

added to the cells. The following day the cells were washed in 5ml TBS (Appendix

1.2) for 10 minutes at room temperature. The washing procedure was repeated twice,

and then the medium was replaced in the culture dishes. The cells were split with

trypsin 24 hours later, plated into 6 well culture dishes and left to grow for 2-3 days.

Individual clones of cells that were grown in cloning rings were transferred into 24 well plates and grown to confluence. Control clones were created by the transfection of the neomycin resistance gene only (ie pRc/CMV). Cells were grown in FAD -f FCS + HICE that was supplemented with 2mg/ml geneticin G418 (Geneticin: Gibco) for H357 cells and 1 mg/ml G418 for the H376 cells and individual clones were expanded for

Materials and Methods

FACS selection.

2.1.3 Growth and Selection of the ay and O4 Positive Clones

On reaching confluence in the 24 well plates individual clones were passaged as

described in Section 2.1.1.1 and transferred to 6 well plates (Falcon). When confluent

the cells were split by trypsin/versene treatment and prepared for FACS scanning as described in Section 2.2.1. 75% of the confluent cells were prepared for scanning while the remaining 25% were subcultured. Positive clones were further passaged and then sorted under sterile conditions as described in Section 2.2.2. Stable expression of the integrin subunits was achieved after 4 sterile sorts. Cells were grown in FAD + FCS + HICE medium supplemented with 0418 as described above. Expression of

«V and 8 4 was checked by flow cytometry about every four weeks.