Lymphoma 6C3HED-OG cells, known from previous work to be susceptible to the effects of guinea pig serum in vivo and dependent upon extrinsic
asparagine for
protein synthesis and growth in vitro, remained for the most part morphologically intact and countable in the electronic cell counter following exposures of 1 and 2 hr to the effects of heated (56 degrees C, 30 min) guinea pig serum injected into the peritoneal cavities of mice in which the
lymphoma cells were growing rapidly; after exposures of 4 and 6 hr the bulk of the -OG cells remained still intact and countable in the cell counter, though by this time a small proportion of them (5 to 12%) proved stainable with
eosin in wet preparations) hence were presumably nonviable. After 12, 16, and 24 hr of exposure, however, the bulk of the -OG cells were either lysed or fragmented, to the extent that they did not register in the cell counter. Morphologic studies of the cells exposed 16 and 24 hr to the effects of heated guinea pig serum in vivo, disclosed that most of the cells then remaining were either frankly necrotic or greatly altered otherwise, marked vacuolation of the cytoplasm being the most conspicuous alteration in cells not yet obviously necrotic. Long before the bulk of the
Lymphoma 6C3HED-OG cells had become conspicuously changed morphologically following exposure to the effects of heated guinea pig serum in vivo, they manifested striking alterations in
protein metabolism, as was disclosed by "pulse" studies with radioactive
valine. For example, the
protein metabolism of -OG cells, as measured by their incorporation of
L-valine-C(14), was sharply curtailed following 15 min of exposure to heated guinea pig serum in vivo, as compared with
valine incorporation by cells labeled immediately after exposure to the guinea pig serum. Following exposure to heated guinea pig serum during 60 min, -OG cells incorporated less than half as much
L-valine-C(14) as did cells labeled immediately after exposure, and the incorporation of
L-valine-C(14) was still less after 120 min of exposure. By contrast,
Lymphoma -RG1 cells, known from previous work to be wholly insusceptible to the effects of guinea pig serum in vivo and independent of need for extrinsic
asparagine for
protein synthesis and growth in vitro, showed no curtailment whatever of
protein synthesis following exposures to the effects of heated guinea pig serum in vivo during periods of 15, 60, and 120 min. Reasons are given for considering the prompt inhibition of
protein synthesis in the
asparagine-dependent -OG cells a direct result of
asparagine-deprivation induced in vivo by the injected guinea pig serum, the L-
asparaginase of which presumably converted the available
L-asparagine of the host to
L-aspartic acid that was not taken up by the -OG cells. The synthesis of
deoxyribonucleic acid by
Lymphoma 6C3HED-OG cells, as measured by the incorporation of thymidme-H(3), determined with the aid of liquid scintillation counting and autoradiography, was also altered by exposure of the
lymphoma cells to the effects of heated guinea pig serum in vivo, though not during exposures of 15 and 60 min; only after an exposure of 120 min did the population of -OG cells incorporate notably less
thymidine-H(3) than did control populations, though after 240 min of exposure the -OG cells incorporated less than one-fifth as much tritiated thymidineas had -OG cells exposed to heated guinea pig serum for 60 min or to heated horse serum for periods up to 240 min. Autoradiographs indicated that
DNA synthesis by -OG cells normally proceeds at an intense level that leads to some 60% of these cells being heavily labeled in autoradiographs at any given time; after exposure to the effects of heated guinea pig serum during 2 and 4 hr in vivo, however, the
lymphoma cells lost their ability to incorporate enough tritiated
thymidine to become heavily labeled, but approximately the same proportion of them (56 to 58%) retained their ability to incorporate sufficient tritiated
thymidine to become lightly labeled. The possibility is considered that the inhibition of
DNA synthesis in the
asparagine-dependent -OG cells exposed to the effects of heated guinea pig serum in vivo may be secondary to the previously manifest inhibition of
protein synthesis. Further, in tests of
ribonucleic acid metabolism of
Lymphoma 6C3HED-OG cells after exposure to the effects of heated guinea pig serum in vivo during periods of 15, 60, 120, and 240 min, the findings indicated that the ability of the
lymphoma cells to synthesize
RNA, as measured by their capacity to incorporate
uridine-5-H(3), remained unaltered during the exposures of 15, 60, and 120 min, but was substantially reduced following 240 min of exposure. The findings are considered in relation to the probability, disclosed in part by previous studies, that heated guinea pig serum brings about its effects upon
Lymphoma 6C3HED-OG cells in vivo by providing active L-
asparaginase in large amounts, which presumably converts the available (extracellular)
asparagine of the host to
aspartic acid, the latter not being taken up by the
lymphoma cells in vivo or in vitro. Hence it seems likely that heated guinea pig serum in this way brings about a state of
asparagine deprivation that is responsible for the sequential metabolic and morphologic alterations that become manifest in
asparagine-dependent
Lymphoma 6C3HED-OG cells following their exposure to the effects of guinea pig serum in vivo, as here described.