Reduction of Melanogenic Activity of Responsiveness to alpha-Melanocyte-Stimulating Hormone during Serial Passage of Melanoma Cells
Hee-Young Park and Barbara A.
Gilchrest
Background: Murine melanoma
cells such as Cloudman S91 or B16 mouse melanoma cells have been
used extensively to study mechanisms involved in pigmentation
because these cells have tyrosinase, the key enzyme in
pigmentation, and produce pigment. We have observed that serial
passaged S91 cells tend to decrease their basal pigment content
and to lose their responsiveness to alpha-melanocyte-stimulating
hormone (alpha-MSH).
Objective: Because this reduction
of melanogenic capacity is a widely acknowledged but virtually
unstudied characteristic of both human and murine melanoma cell
lines in culture, we wished to document and quantify the
phenomenon.
Methods: Commercially attained S91
melanoma cells were serially passaged. Basal pigmentation as well
as alpha-MSH responsiveness and expression of protein kinase
C-beta (PKC - beta) were assessed.
Results: S91 cells progressively
lost their basal pigmentation under standardized conventional
conditions of culture, from an initial melanin content of 20 ± 4
pg/cell content to 12 ± 5 pg/cell within 70 population doublings
(16 passages) and to 4.5 ± 6 pg/cell, a level or below the
detectable level of our melanin assay, by 110 population
doublings (28 passages). When responsiveness to alpha-MSH was
assessed, a 6-day treatment with 10-6
M alpha-MSH initially induced the pigment content four-fold from
20 ± 4 to 82 ± 2 pg/cell. PKC-beta expression was readily
detected by immunofluorescence in early passage pigmented cells,
but not in late passage non pigmented cells.
Conclusion: These results confirm
that while murine melanoma cells are a useful model system for
pigmentation studies, it is important to monitor changes in the
cells' basal abilities to pigment and to respond to exogenous
pigment-inducing factors. They further suggest that factors in
the culture environment or the internal milieu of melanoma cells
exposed to continuous mitogenic stimulation inhibit
melanogenesis. One candidate mechanism is down regulation of
PKC-beta.
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