466
CLIFFORD GROBSTEIN
Behaviorally as well as morphologically, cells in culture tend to become
increasingly alike. It is true that long-term cultured cell strains show
persistent behavioral differences, but the relation of these to the bedavioral differences of cell types in vivo usually is not direct. Behavioral
criteria, like morphological ones, tend to fail just when they are needed
most—when differentiation is suspected of being present but in early,
attenuated, or atypical form.
Increasingly, therefore, interest has focused on detecting changes at
the molecular level, in the hope either that these may be more persistent
or that their detection may provide more sensitive tests than behavioral
and morphological observations. Underlying this approach is the
assumption that differentiated states involve specialized metabolism,
particularly specific biosynthetic pathways which produce the particular products whose effects are seen in differentiated structure and
function. Detection and measurement of product, of characteristic
enzymes, of uptake of unique substrates—all of these may be used as
markers of the differentiative process, and of the presence of differentiated cells. Where sufficient amounts of cells or tissues are available,
ordinary biochemical procedures have been used. Where the amounts
available are small, or where differentiation may exist in only a small
fraction of the cells, bulk methods must give way to micromethods.
Microanalysis, cytochemistry, autoradiography and immunochemistry
each have their place in recognizing differentiated states in cultured
cells. When, for example, the product is an effective antigen, as is the
case with myosin of muscle, fluorescent antibody techniques have
proven extremely sensitive in detecting the beginnings of specific
synthesis (Engel and Horvath, 1961). Where synthesis involves a relatively specific substrate, as with the sulphated mucopolysaccharides of
cartilage matrix, uptake of the radiologically labelled substrate and its
detection chemically (Lash, Holtzer and Whitehouse, 1960) or autoradiographically is applicable.
Caution needs to be exercised, however, in applying these newer,
more sensitive methods to the recognition of cytodifferentiation.
Sensitivity and high resolution, as is so frequently true, are here
achieved through narrowing the field of observation. Narrow concentration needs to be balanced by simultaneous application of possibly
less sensitive but broader range procedures. The occurrence in vitro of an
enzyme characteristic of a specialized cell in vivo, e.g. arginase in liver
cells, is not diagnostic of a particular differentiation if that enzyme may
arise and can be detected in cultured cells from sources not normally
producing it (Hillis and Bang, 1962). Nor does the demonstration of one
biosynthesis characteristic of a cell type necessarily imply that all
properties of the cell type are being displayed. Although the various
CLIFFORD GROBSTEIN
Behaviorally as well as morphologically, cells in culture tend to become
increasingly alike. It is true that long-term cultured cell strains show
persistent behavioral differences, but the relation of these to the bedavioral differences of cell types in vivo usually is not direct. Behavioral
criteria, like morphological ones, tend to fail just when they are needed
most—when differentiation is suspected of being present but in early,
attenuated, or atypical form.
Increasingly, therefore, interest has focused on detecting changes at
the molecular level, in the hope either that these may be more persistent
or that their detection may provide more sensitive tests than behavioral
and morphological observations. Underlying this approach is the
assumption that differentiated states involve specialized metabolism,
particularly specific biosynthetic pathways which produce the particular products whose effects are seen in differentiated structure and
function. Detection and measurement of product, of characteristic
enzymes, of uptake of unique substrates—all of these may be used as
markers of the differentiative process, and of the presence of differentiated cells. Where sufficient amounts of cells or tissues are available,
ordinary biochemical procedures have been used. Where the amounts
available are small, or where differentiation may exist in only a small
fraction of the cells, bulk methods must give way to micromethods.
Microanalysis, cytochemistry, autoradiography and immunochemistry
each have their place in recognizing differentiated states in cultured
cells. When, for example, the product is an effective antigen, as is the
case with myosin of muscle, fluorescent antibody techniques have
proven extremely sensitive in detecting the beginnings of specific
synthesis (Engel and Horvath, 1961). Where synthesis involves a relatively specific substrate, as with the sulphated mucopolysaccharides of
cartilage matrix, uptake of the radiologically labelled substrate and its
detection chemically (Lash, Holtzer and Whitehouse, 1960) or autoradiographically is applicable.
Caution needs to be exercised, however, in applying these newer,
more sensitive methods to the recognition of cytodifferentiation.
Sensitivity and high resolution, as is so frequently true, are here
achieved through narrowing the field of observation. Narrow concentration needs to be balanced by simultaneous application of possibly
less sensitive but broader range procedures. The occurrence in vitro of an
enzyme characteristic of a specialized cell in vivo, e.g. arginase in liver
cells, is not diagnostic of a particular differentiation if that enzyme may
arise and can be detected in cultured cells from sources not normally
producing it (Hillis and Bang, 1962). Nor does the demonstration of one
biosynthesis characteristic of a cell type necessarily imply that all
properties of the cell type are being displayed. Although the various