In vivo Expression Analysis of Severe Combined

3.2.11 In vivoExpression Analysis of Severe Combined Immunodeficiency Mice (SCID)

Figure 3.67: Transplantation of Infected Breast and non Breast Cells into SCID/bg Mice Mice were housed in microisolator cages, fed only autoclaved food and water, and handled only with gloves.

Female mice that weighed between 17 and 20 g were anaesthetised with ketamin (Ketalar 1%, 0.2 ml/20 g mouse i.p). Antibiotic (Borgal^R24%) was added to the drinking water on the day of implantation. For the implantation, a small area of skin was shaved (~2x2 cm) and disinfected. A small incision was made in the skin and an oestrogen pellet pushed into it. Depending on the size of the wound, the cut was either sewn together (by a veterinarian) or simply pushed together. 24 hours later 1x10⁷cells that had been previously mixed with Matrigel were injected into the mammary fat pad. After a 5-8 week incubation period, the mice were killed and the tumour immediately removed from the surrounding tissue.

3.2.11.2 Protein Expression Analysis

Protein was extracted from the tumours of 3 mice per tumour type. Tumours were homogenised on ice in lysis buffer. The tumour extracts were then treated with a cocktail of protease inhibitors [PMSF (0.2 mM) and leupeptin (5 µg/ml)] in conjunction with a heat inactivation step, just before analysis, in order to reduce high levels of endogenous ß-galactosidase activity that had been previously observed in tissue samples (Shaperet al., 1994). Every human breast tumour showed high levels of ß-galactosidase expression from the WAP-BAGgal hybrid vector when compared to the parental BAGgal (Fig. 3.68).

In vivobreast tumours

MCF7 ZR-75-1 T-47D

0 10 20 30

Mock BAGgal WAP-BAGgal

foldbackground

In vivonon-breast derived human tumours

EJ Panc-1

0 1 2 3 4

Mock BAGgal WAP-BAGgal

foldbackground

a)

b)

Figure 3.68: Analysis of ß-galactosidase Expression in Human Breast and non-Breast Tumour Cell Xenografts in SCID/bg Mice

a) Expression of ß-galactosidase in human breast cell derived tumours in SCID/bg mice.

10 µg of protein that had been previously been extracted from tumours that had arisen after implantation of tumour cells was used for a quantitative galactosidase assay (Galactolight, Perkin Elmer). The expression of galactosidase is measured in relative light units after a chemoluminescent substrate is added. The background ß-galactosidase activity was set to a value of 1 and the expression of the vectors then calculated in relationship to the background. High levels of expression could be seen from the hybrid WAP-BAGgal construct in MCF7, ZR-75-1 and T-47D cells. Expression from the BAGgal vector was lower by comparison, with no expression being observed in T-47D cells.

b) Expression of ß-galactosidase in human non-breast cell derived tumours in SCID/bg mice

The experiment was performed in the same manner as described above for human breast cell derived tumours.

Expression of the hybrid WAP-BAGgal vector could not be seen in both non-breast tumour cell lines examined.

High levels of expression from the parental BAGgal vector could be seen in EJ cell derived tumours. No significant expression could be seen in Panc-1 cell derived tumours. The results are shown as the mean of three independent experiments (error bars show the the standard error of the mean).

The T-47D cells showed expression levels from the WAP-BAGgal hybrid vector 21.2 times that of the background, whereas the BAGgal levels were only 1.4 times that of the empty cells.

Expression could also be seen in both ZR-75-1 as well as MCF7 cells to a lesser exetent, being 27.2 and 10.4 fold background respectively. Expression from the parental BAGgal vector could also be seen in MCF7 and ZR-75-1 cells, but was always at lower levels than those observed for the hybrid vector. The high levels of expression from WAP-BAGgal could also be shown histologically in both T-47D (Fig. 3.69a) and ZR-75-1 tumours after fixation and staining (see 2.5.3.2.2).

In the non-breast tumours cell lines examined, EJ and Panc-1, no significant expression could be seen from the WAP- BAGgal hybrid vector. Low levels of expression were seen in EJ and Panc-1 cells infected with the parental BAGgal being 2.5 and Panc-1.45 fold background respectively.

Histological analysis confirmed these results (Fig. 3.69b)

Figure 3.69: Histological Analysis of ß-galactosidase Expression in non-Breast Tumour Cell Xenografts in SCID/bg Mice

a) X-gal staining of breast cell derived tumours.

A tumour derived from implanted WAP- BAGgal infected T-47D cells was dissected and cut into small pieces beforebeing fixed. The fixation was made in 4% paraformaldehyde for 24 hours at 4°C. The tissue was then stained with x-gal (2.5.3.2.2) before being embedded in paraffin and photographed. Blue staining indicates areas of ß-galactosidase expression. Similar results were also obtained for ZR-75-1 tumours (not shown).

b) X-gal staining of non-breast cell derived tumours.

The experiment was performed in the same manner as b). No expression, normally indicated by blue areas, can be seen in the EJ cell derived tumour.

3.2.11.3 RNA Expression Analysis

RNAanalysis was made to show the expression levels of ß-galactosidase in the tumours arising from breast and non-breast derived cells. Northern blot analysis was performed as it not only indicates the levels of expression but also allows the length of the transcript to be determined.

Total RNA was isolated from a tumour from 1 of the 4 mice per construct per cell and run over a denaturing formaldehyde gel (and, after being transferred to a nylon membrane via capillary blotting, was hybridised to a radioactively labelled 0.4 kb ß-galactosidase specific fragment. The 7 kb fragment that could be seen in all human breast cell derived tumours in both the hybrid WAP- BAGgal as well as the parental BAGgal vector (Fig. 3.70, lanes 1, 2, 3, 6, 7 and 8) corresponds to the full length vector transcript. The strongest ß-galactosidase expression could be observed in those human breast cell derived tumours that had been infected with the hybrid WAP-BAGgal vector before implantation. The ß-galactosidase expression could not be detected in bladder carcinoma cell derived tumours (EJ, lane 4) where the MLV promoter showed higher levels of expression than the WAP promoter (EJ, lane 9). However, in pancreatic cell derived tumours (Panc-1, lanes 5 and 10), no expression could be detected from either the WAP-BAGgal or BAGgal vectors. There was no expression in non- infected tumours (lanes, 11, 12, 13, 14, and 15). The same membrane was then stripped and hybridised to a radioactively labelled 1.2 kb ß-actin specific fragment. It shows the presence of similar quantities of RNA in each lane.

Figure 3.70: Northern Blot Analysis of Human Tumours from SCID/bg Mice

20µg of total RNA was separated on a denaturing formaldehyde gel and transferred to a nylon membrane using capillary blotting. The membranes were then hybridised to anα³²P labelled 0.4 kb ß-gal specific fragment (from pBAGgal afterMluI digestion) and, following documentation and stripping, also to anα³²P labelled 1.2 kb ß-actin fragment [from pAL41 (Alonsoet al., 1986) afterPstI digestion]. Lane 1, RNA from a WAP-BAGgal infected ZR-75-1 tumour; lane 2, RNA from a WAP-BAGgal infected T-47D tumour; lane 3, RNA from a WAP-BAGgal infected MCF-7 tumour; lane 4, RNA from a BAGgal infected EJ tumour; lane 5, RNA from a WAP-BAGgal infected Panc-1 tumour; Lane 6, RNA from a WAP-BAGgal infected ZR-75-1 tumour; lane 7, RNA from a BAGgal infected T-47D tumour; lane 8, RNA from a BAGgal infected MCF-7 tumour; lane 9, RNA from a BAGgal infected EJ tumour; lane 10, RNA from an WAP-BAGgal infected Panc-1 tumour. Lane 11, RNA from infected infected ZR-75-1 tumour; lane 12, RNA from infected T-47D tumour; lane 13, RNA from a non-l infected MCF-7 tumour; non-lane 14, RNA from a non-non-l infected EJ tumour; non-lane 15, RNA from an non- infected Panc-1 tumour.