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tumor occurrence in 50% of animals in any two untreated groups in two different
experiments may be different (for example 60 weeks versus 50 weeks). Thus, each
experiment always has an untreated control group for assessment of the effect of a
particular treatment. In all the transplantation experiments described here, three
different donors were used for each experiment with equal representation in the
different groups.
In experiment 1 there were two groups of mice. Group 1 comprised untreated
control mice and group 2 comprised mice that received Silastic tubing containing
50 µg of estradiol-17
β
and 20 mg of progesterone for the period of weeks 2 to 4
after transplantation. The Silastic tubings were implanted subcutaneously dorsally
and removed after the 2-week period. Mice were palpated weekly until 45 weeks
after transplantation. At that time, the mammary fat pads free of palpable tumors
were either collected for the preparation of epithelial cell pellets and frozen or pre-
pared as whole mounts. This experiment was repeated identically and designated
experiment 1A. Additionally, transplants were collected between weeks 8 and 16
after transplantation to assess proliferation activity by bromodeoxyuridine (BrdU)
immunohistochemistry. The mice were injected intraperitoneally with BrdU 2
hours before tissue collection and the samples were processed as described previ-
ously [17]. A total of 500 cells were counted from each fat pad under ×10 magni-
fication. There were four fat pads in each treatment group. In experiments 1 and
1A, a total of 66 transplants per group were assessed for tumorigenic potential.
Experiment 2 was started 12 months after experiment 1A and addressed the
question of whether the developmental stage of the mammary epithelium in-
fluenced the response to the short-term hormone exposure. In this experiment
there were four groups of mice. Groups 1 and 2 were identical to the groups in
experiment 1. Thus, epithelial cells were actively proliferating as the ducts were
filling the fat pad. The proliferation index at this stage was about 8% [11]. Group
3 comprised mice that received the hormone treatment at 23 to 25 weeks after
transplantation at a period when the mammary epithelium had filled the fat pad
and proliferation was in a steady state but tumors were starting to appear. We
included a positive control as group 4, in which the mice were exposed to a 5 mg
pellet of tamoxifen between weeks 11 and 24. Previous experiments had shown
that lifetime exposure to tamoxifen prevented the development of mammary tu-
mors in this model system [18]. The mice were palpated weekly until 50 weeks
after transplantation.
Experiment 3 tested whether pretreatment of the recipient mice with hor-
mones could provide a protective effect on mammary epithelium that had not
been directly exposed to the added hormone exposure. In this experiment there
were four groups of mice. Groups 1 and 2 were as in experiment 1, in which the
duct fragments were transplanted into 3-week-old mice and estrogen/progesterone
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