ANÁLISIS COMPARATIVO

An estrogen receptor negative human breast tumour cell line, MDA-MB-435S, was used for further in vitro _{analysis experiments. Here, the pLXSN based pLXPCWAPEGFP retroviral} vectorwas used. The reporter gene in this vector is EGFP instead of ß-galactosidase and carries a similar modification of the 3' LTR as in pWAP-BAGgal. It should also undergo reverse transcription in a similar manner leading to expression being controlled by the WAP NRE after infection of target cells. As it is well known (Lesoon-Wood et al_{., 1995) that the MDA-MB-} 435S cells are highly metastatic, the use of EGFP as a marker gene should allow easier detection of expression in an in vivo _{mouse model where these cells have been transplanted and} metastases allowed to develop.

MDA-MB-435S as well as T-47D breast tumour cells were infected with either pLXPCWAPEGFP or pLXSNEGFP. As it had already been shown that the WAP NRE was active in T-47D cells they were used as a positive control in this series of experiments.

MDA-MB-435S and T-47D cells were cultured in a monolayer system and then infected with with either the hybrid LXPCWAPEGFP or the parental LXSNEGFP vector. As in the infection experiments that have been previously described, the recombinant retroviral particles were here also pseudotyped with the VSV-G protein in order to obtain enough clones to establish a population (>50 clones) after G418 selection (MDA-MB-435S, 400 µg/ml: T-47D, 200 µg/ml). The stably infected cells were then seeded out in a 3D cell culture system and stimulated for 3 days with prolactin (3 µg/ml), insulin (3 µg/ml) and dexamethasone (10-6M). The expression of

the enhanced form of the green fluorescent protein (EGFP) was examined using fluorescence microscopy. Expression from the WAP promoter could be seen in both infected cell lines after hormone stimulation. Here, the expression from the BAGgal vector was seen to be much stronger than the WAP expression levels in MDA-MB-435S cells (Fig. 3.62). It was also observed that infected MDA-MB-435S cells that had grown to high confluency exhibited higher levels of expression than non-confluent cells. This supports the theory that a complex structure of cells is important for the expression from the WAP promoter.

Figure 3.62: EFGP Expression in Transduced MDA-MB-435S Human Breast Tumour Cell Lines

The breast tumour cell lines MDA-MB-435S and T-47D were stably infected with either the hybrid LXPCWAPEGFP or the parental LXSNEGFP vector and cell populations established. Infected cells that had been cultured in matrigel were examined using fluorescent microscopy after 3 days hormone treatment [prolactin (3

µg/ml), insulin (3 µg/ml) and dexamethasone (10-6 M)].

Non-infected MDA-MB-435S cells with stimulation (panel 1) show no green EGFP expressing cells. LXPCWAPEGFP infected cells with stimulation (panel 2) show many green, EGFP expressing cells. MDA-MB- 435S cells infected with the parental LXSNEGFP vector show strong EGFP expressing cells (panel 3). 400x magnification.

3.2.10.6 S1-Analysis

To accurately quantitate at the transcriptional level expression from the WAP-BAGgal infected cells grown under different conditions, S1 analyses performed.

Total RNA was prepared from stably infected T-47D and ZR-75-1 human breast cell lines that had either been grown on collagen and stimulated for 3 days or grown in monolayer and not stimulated at all. Non-infected T-47D and ZR-75-1 cells were also treated in the same manner and RNA isolated for use as a negative control. Primary human breast tumour cells obtained from a biopsy were also examined in this experiment. Both infected and uninfected cells were stimulated for 3 days before the RNA extraction was made. These RNAs were then hybridised to a γ32P-end-labelled BsaI DNA fragment from the recloned hybrid MMTV-BAGgal vector (pMMTVProCon). The same probe was used here as was used to determine the transcriptional start site of the MMTV-BAGgal recloned vector. However, as the sequences upstream of the R region differ between the two vectors, no conclusion can be made here as to the start site of the WAP-BAGgal transcript.

A clear induction of the signal could be observed in RNA derived from cells grown on collagen in the presence of hormones (lanes 1, 2 and 7) compared to that from cells grown in monolayer in the absence of the hormones (lane 8). This also shows that the start of transcription is located in the 5’LTR, presumably under the control of the WAP promoter.

WAP GAPDH 90 bp 200 bp a) b) LacZ Integrated WAP-BAGgal mRNA SD Bsa_I S1 probe 89 nt WAP NRE

Figure 3.63: S1 Nuclease Protection Analysis

a) Diagram of the S1 probe used. The probe was isolated after a Bsa_{I digest of the recloned hybrid MMTV-}

BAGgal plasmid pMMTVProCon. The protected fragment after S1 digestion was 90 nucleotides.

b) Total RNA was isolated from a stably infected population of hybrid WAP-BAGgal infected cells grown on collagen and stimulated with hormones for 3 days before RNAisolation. 40 µg total RNA was used per reaction.

RNA was hybridised against aγ32P-end-labelledBsaI LTR DNA fragment as previously described (Günzburget

al_{., 1986). After S1 digestion, a protected fragment of a 90 bp was obtained, as determined with Phosphor imager}

software (Molecular Dynamics). RNA from WAP-BAGgal infected 1° human breast tumour cells grown on collagen in the presence of hormones (lane 1) or non-infected 1° cells (lane 2). The RNA of infected, hormone treated T-47D cells (lane 3) gives a stronger signal than that of infected, hormone treated ZR-75-1 cells (lane 7) and infected, non-stimulated cells T-47D and ZR-75-1 cells (lanes 4 and 8 respectively). No signal was obtained either from the RNA derived from non-infected cells cultured on collagen with hormonal stimulation or from

monolayer culture in the absence of hormones (lanes 2, 5, 6, 9 and 10).Hae_{III-digested pBR322 was used as a}