Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.

Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.

Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions.

We have previously described a method for producing recombinant methionyl bovine PRL (Met-bPRL), which is as bioactive as the authentic hormone in the Nb2 cell lactogen bioassay; in contrast, a Met-bPRL variant lacking tyrosine 28 was essentially devoid of bioactivity. In the present study we have investigated this loss of bioactivity at the molecular level by determining the bioactivities of a number of Met-bPRL variants engineered to contain specific changes in their primary structures. It was found that the presence of tyrosine per se at the 28 position in Met-bPRL was not essential for high bioactivity, since Met-bPRL variants prepared by replacing tyrosine 28 with other amino acids (arginine, phenylalanine, alanine, and histidine) still had substantial bioactivity (40-74% that of Met-bPRL). Neither was the loss of bioactivity related to a shift in the relative positions of conserved histidines 27 and 30; in fact, histidine 27 was found not to be essential for the bioactivity of the hormone. The loss of bioactivity after deletion of tyrosine 28 from Met-bPRL appears to be related to the removal of an amino acid from the middle of a putative helix (no. 1) rather than to the loss of a residue specific to lactogen function. This suggestion is supported by the finding that Met-bPRL variants obtained by deletion of selected single amino acids from center domains of putative helix 2, 3, or 4 were also essentially devoid of bioactivity. It is speculated that this lack of bioactivity reflects an inability of the proteins to assume a native conformation.

Bioactive recombinant methionyl bovine prolactin: structure-function studies using site-specific mutagenesis.

A method has been developed for the extraction from transformed Escherichia coli cells of methionyl bovine PRL (met-bPRL) in a relatively pure form. While the extracted met-bPRL was as reactive as the native hormone with respect to polyclonal anti-bPRL antibodies, its bioactivity, as measured by the Nb2 lactogen in vitro bioassay, was relatively low. The bioactivity of the met-bPRL could be increased to the same order as that of the native hormone by treatment with a mixture of oxidized and reduced thioredoxin. A number of variant met-bPRLs containing specific amino acid changes have been generated by site-specific mutagenesis. The changes involved the substitution (or deletion) of some of the conserved amino acids in bPRL by the different amino acids present at the corresponding positions in the related, but nonlactogenic bovine GH. Nine mutants containing single amino acid changes had bio- and immunoactivities of the same order as those of met-bPRL. One mutant, which incorporated two of the single amino acid changes (serine 62 to threonine and threonine 65 to alanine), had immunoactivity approximating that of met-bPRL but much lower bioactivity (45%). A further mutant, generated by the deletion of tyrosine 28, had essentially no bioactivity although it could not be distinguished immunologically from met-bPRL or bPRL. The findings are discussed in the light of the putative three-dimensional PRL structure and current hypotheses which seek to relate specific regions of PRL to lactogenic activity. It appears that the first putative alpha-helix of bPRL is important for the binding and mitogenic activity of the hormone.

Presence of prolactin-like immunoreactivity and bioactivity in rat spinal cord.

Pursuant to our identification of prolactin-like immunoreactivity (PLI), widely distributed in rat brain, the spinal cord was examined for the presence of this pituitary-hormone-like protein. PLI was present in all spinal cord extracts examined and averaged 500 +/- 53 pg/mg protein. Hypophysectomy, causing a fall in serum prolactin to undetectable levels, was not associated with any change in levels of PLI in spinal cord. Recovery of rat prolactin standards added to spinal cord homogenates was 97.6 +/- 3.9%. When increasing concentrations of spinal cord extract were assayed in a prolactin radioimmunoassay, displacement of rat 125I-Prolactin from antiserum was parallel to that displacement produced by increasing concentrations of rat anterior pituitary standards. Upon subjection to gel permeation chromatography, the elution profiles of immunoreactive prolactin from spinal cord were different from the profiles of anterior pituitary prolactin. In addition to an immunoreactive prolactin peak eluting with pituitary prolactin, spinal cord extracts showed a large void volume peak and late eluting low-molecular-weight materials not seen with anterior pituitary. In the Nb2 lymphoma cell assay, all spinal cord extracts demonstrated prolactin-like bioactivity with a bioactivity/immunoreactivity ratio of 1.05 +/- 0.13. We conclude: (1) PLI, widely distributed in rat brain, is also present in spinal cord; (2) spinal cord prolactin levels are independent of levels in pituitary and peripheral circulation; (3) this immunoreactive prolactin is bioactive, and (4) differing gel permeation chromatographic elution profiles indicate that there may be some molecular differences between pituitary and spinal cord prolactin.

Extrahypothalamic brain prolactin: characterization and evidence for independence from pituitary prolactin.

Prompted by reports of immunohistochemical localization of a prolactin-like immunoreactivity (PLI) within the rat brain, a study was undertaken to define the immunologic and biologic characteristics of this material in extrahypothalamic regions of the rat brain. Ninety-seven percent recovery of rat prolactin standard, added to homogenates of brain parts, insured that neuronal tissue did not interfere with the radioimmunoassay for rat prolactin. PLI was consistently found in the cerebellum, thalamus, brainstem (pons-medulla), hippocampus, cerebral cortex and caudate. Examination of the elution profile of each of the extrahypothalamic brain parts from Sephadex G-75 columns showed that, although a small amount of brain PLI elutes in the vicinity of the anterior pituitary prolactin marker, the bulk of brain-based PLI migrates with the void volume and as late eluting, low molecular weight material. While increasing amounts of brain extracts progressively displaced more 125I-prolactin from antibody binding, the displacement curve was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Extracts of various brain parts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoreactivity ratio for some of the brain parts was significantly lower than that for pituitary prolactin. Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in extrahypothalamic brain parts, indicating that brain PLI is regulated independently of pituitary prolactin and of circulating serum prolactin levels.

Prolactin-dependent mitogenesis in Nb 2 node lymphoma cells: effects of immunosuppressive cyclopeptides.

Prolactin (PRL)-stimulated ornithine decarboxylase (ODC) activity and subsequent proliferation are inhibited by the cyclopeptides cyclosporine (CsA) and didemnin B (DB) in Nb 2 node lymphoma cells. Similar concentrations of these agents also inhibit 125I-PRL binding, suggesting that their inhibitory effects on these PRL-dependent physiologic responses are mediated at least in part at the level of PRL receptor interactions. The phorbol ester TPA stimulated ODC activity and [3H]thymidine incorporation to 54% and 31% that of a near-optimal mitogenic concentration of PRL (10 ng/ml), suggesting that mitogenesis in these cells is coupled to some degree to the activation of protein kinase C (PKC). The calcium ionophore A23187 increased ODC activity only slightly and actually decreased [3H]thymidine incorporation to a value below the "cells only" controls. The addition of TPA plus A23187 did not further enhance the effects of TPA to elevate ODC activity and [3H]thymidine incorporation. However, A23187 significantly elevated PRL-stimulated ODC activity with a subsequent inhibition of [3H]thymidine incorporation, suggesting a block of entry into S phase. Both cyclopeptides decreased the elevation of ODC activity in G1 phase of cell cycle in response to PRL, suggestive of a site of action for these agents in early G1, a conclusion compatible with their ability to inhibit PRL binding to these cells. Addition of CsA or DB 2 hr after PRL had no effect on PRL-stimulated ODC activity detectable at 6 hr, but addition of either as late as 6 hr still affected the extent of mitogenesis. This is in line with the requirement for PRL to be present in the culture medium for a minimum of 3 to 6 hr to invoke a maximal effect on mitogenesis. Addition of either cyclopeptide after the cells were in S phase had no effect on the extent of [3H]thymidine incorporation. An inhibitor of the cyclooxygenase pathway (indomethacin) enhanced both PRL-stimulated ODC activity and proliferation, whereas inhibition of the lipoxygenase pathway by NDGA attenuated only proliferation, suggesting that in Nb 2 cells, products of the lipoxygenase pathway may contribute to the mechanism of PRL-stimulated mitogenesis. Because Nb 2 lymphoma cells were derived from estrogenized rats, estrogen was tested as a mitogen. By itself it was not mitogenic, but in conjunction with PRL, estradiol-17 beta elevated the ODC response and inhibited proliferation. Inhibitors of PKC known to have minimal effects on RNA synthesis, quercetin and gossypol, totally inhibited both the elevations of ODC activity and [3H]thymidine incorporation in response to PRL in Nb 2 lymphoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

An inhibitor of ornithine decarboxylase in lactogen-deprived Nb2 node rat lymphoma cells.

A previous study has shown that the activity of ornithine decarboxylase in cultured Nb2 node rat lymphoma cells falls to undetectable levels when cells become quiescent following incubation in lactogen (prolactin)-deficient medium. In the present study, it was found that addition of extracts of the lactogen-deprived, quiescent cells to extracts of log-phase cells markedly reduced the ornithine decarboxylase activity of the latter, the inhibitory activity being proportional to the amount of quiescent cell extract added. Evidence is presented that the ornithine decarboxylase-inhibitory activity in the quiescent cell extracts is due to an antizyme-like, polypeptide factor with an Mr of approx. 28,000. The activity of the inhibitor appears to be directed rather specifically against ornithine decarboxylase, since the activities of S-adenosylmethionine decarboxylase, thymidine kinase and uridine kinase were not affected. The Nb2 cell ornithine decarboxylase inhibitor may have an important role in modulating the cellular levels of ornithine decarboxylase as they change in response to the withdrawal and restoration of extracellular mitogenic lactogens.

Prolactin stimulation of ornithine decarboxylase and mitogenesis in Nb2 node lymphoma cells: the role of protein kinase C and calcium mobilization.

The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in combination with calcium ionophores has been shown to bypass the requisite antigen- or lectin-induced signal for lymphocyte mitogenesis. This suggests that protein kinase C activation and calcium mobilization may be early events required for lymphocyte proliferation. Therefore, the relationship(s) of protein kinase C activation and calcium mobilization to ornithine decarboxylase induction and cellular proliferation were examined in a rat node lymphoma cell line (Nb2) which is dependent upon prolactin (PRL) for mitogenesis. TPA enhanced PRL-stimulated Nb2 node lymphoma cell ornithine decarboxylase induction and [3H]thymidine incorporation. Addition of a calcium ionophore (A23187) to cultures containing TPA plus PRL increased ornithine decarboxylase above PRL alone or PRL plus TPA but inhibited proliferation compared to the PRL plus TPA regimen. Exposure of cells to TPA or TPA plus A23187 increased [3H]thymidine incorporation in a similar manner to that demonstrated for low-dose PRL. However, optimal concentrations were only 20-25% as effective as mitogens as was optimal PRL. Protein kinase C and calmodulin antagonists inhibited PRL-stimulated ornithine decarboxylase induction and proliferation. Ca2+ chelation or cation channel antagonism inhibited both PRL-stimulated responses. The cyclic AMP analogue, 8Br-cAMP, inhibited PRL-stimulated ornithine decarboxylase activity as well as cellular proliferation processes assessed by [3H]thymidine incorporation. Finally, tumor-promoting phorbol esters inhibited 125I-rPRL binding. These data strongly suggest that protein kinase C activation and calcium mobilization are requisite events for PRL-stimulated ornithine decarboxylase induction and cellular proliferation in Nb2 node lymphoma cells. An additional component that is linked to alterations in K+ channeling is also implicated. These data support a role for protein kinase C in PRL-coupled mitogenesis. However, other critical Ca2+ and/or ion-induced events are also required.

Cultured Nb rat lymphoma cells in endocrine and cancer research.

This review outlines the establishment, properties, and use of two lines of cultured Nb rat lymphoma cells. The cultured cells have retained important properties of the cancers of origin, such as dependency on prolactin for growth and a high sensitivity to antineoplastic Vinca alkaloids. The cultures have been useful for defining the hormonal dependency of the lymphomas in the animal and for studying the progression of the lymphomas from hormonal dependency to autonomy. A new, specific and highly sensitive in vitro bioassay for lactogenic hormones has been developed from one of the cultures. The use of the lymphoma cell cultures has revealed unsuspected pharmacological differences between the closely related, clinically useful antineoplastic Vinca alkaloids, vinblastine and vincristine. The lymphoma cell cultures are also useful tools for studying biochemical, cell cycle related events which follow the mitogenic stimulation of cells by a polypeptide growth factor.

Hypothalamic prolactin: characterization by radioimmunoassay and bioassay and response to hypophysectomy and restraint stress.

Prompted by immunohistochemical reports of prolactin-like immunoreactivity in cell bodies within the rat hypothalamus, a study was undertaken to quantitate the immunologic and biologic activity of this material. Hypothalamic concentrations of prolactin-like immunoreactivity averaged 402 +/- 23 pg/mg of protein (n = 30). 97% recovery of rat prolactin standards added to homogenates of hypothalamus insured that neuronal tissue, as prepared for these studies, did not interfere with the radioimmunoassay of rat prolactin. Examination of the elution profile from Sephadex G-75 columns of the prolactin-like immunoreactivity in hypothalamic extracts showed that the majority of hypothalamic prolactin-like substance was of a larger molecular size than pituitary prolactin. While increasing amounts of brain extract progressively displaced more I125 prolactin from antibody-binding sites, the displacement curve produced by adding hypothalamic extract was not parallel to that produced by the addition of increasing amounts of anterior pituitary prolactin standards of rat origin. Hypothalamic extracts from hypophysectomized animals, analyzed for biologic activity in the Nb2 lymphoma cell assay, revealed prolactin-like bioactivity, but the bioactivity/immunoactivity (B/I) ratios for hypothalamic extracts were significantly lower than the B/I ratios for pituitary prolactin (0.71 +/- 0.04 for pituitary, vs. 0.19 +/- 0.06 in the hypothalamus; p less than 0.001). Hypophysectomy, which led to the expected fall in serum prolactin to undetectable levels, and restraint stress, which resulted in a statistically significant 4-fold rise in serum prolactin, caused no change in prolactin concentrations in the hypothalamus, indicating that brain prolactin-like substance is regulated independently of pituitary prolactin and circulating serum prolactin levels.

Vinblastine and vincristine--growth-inhibitory effects correlate with their retention by cultured Nb 2 node lymphoma cells.

Vinblastine (VLB) and vincristine (VCR) have been compared with respect to their inhibitory effects on the growth of cultured Nb2 node rat lymphoma cells and their binding and release by these cells. When examined over a wide concentration range, the two alkaloids were found to be almost equally effective in inhibiting culture growth, providing they were continuously present in the incubation medium. A striking difference was found, however, when the cells were incubated for short periods with the alkaloids and then restored to alkaloid-free medium for further incubation. Cells treated with VCR (5 X 10(-9) g/ml) for 3 h and washed free of extracellular alkaloid failed to resume normal proliferation during a subsequent 48 h incubation period in alkaloid-free medium. In contrast, cells treated with VLB at a higher concentration (5 X 10(-8) g/ml) and for a longer time (7 h), rapidly resumed growth in alkaloid-free medium. Binding studies, using (3H)VLB and (3H)VCR, showed that when cells were incubated continuously with the alkaloids at a given concentration, they bound equal amounts of the drugs. When labelled cells were resuspended in alkaloid-free medium, VLB was released very readily by the cells, whereas VCR was tenaciously retained. The findings indicate that the growth-inhibitory effects of VLB and VCR are related to the amount of alkaloid associated with the cells and that the long-lasting effects of VCR, relative to VLB, are very likely due to the retention of VCR by the cells when the extracellular alkaloid levels decline. The study also suggests that, in the treatment of malignancies, VLB could be more effective if its extracellular levels were maintained, e.g. by continuous administration.

Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line.

PRL and other lactogenic hormones are potent mitogens in a lymphoma cell line derived from a lymph node of an estrogenized Noble (Nb) rat. The present study demonstrates that these cells (designated Nb2 node) possess receptors that bind only lactogenic hormones. There are approximately 12,000 receptor sites per cell. The kinetics of binding of [125I]iodo-PRL exhibited by Nb2 lymphoma cells is unusual in that PRL binding follows a biphasic pattern. Binding of [125I]iodo-PRL reaches a maximum in 1 h at 37 C, followed by a rapid decline. This pattern was not observed if binding was carried out in the presence of chloroquine, a lysosomotropic agent, or if cell homogenate was used for binding. We also examined the relationship between receptor occupancy and the magnitude of PRL response in these cells. Maximal growth stimulation by PRL occurs when only 35% of the maximal binding of PRL is achieved, suggesting that a majority of the PRL binding sites may be spare receptors. This study also revealed that the dissociation constant (Kd) of the PRL receptors in Nb2 cells is 75 pM, approximately 20-fold higher than that of the receptors in other cell types. PRL at 6 pM produces a half-maximal growth response in the Nb2 cells. Antibodies against the PRL receptors are able to abolish the PRL-induced proliferation of Nb2 cells. In the absence of PRL, these antibodies alone can induce proliferation of Nb2 cells, mimicking the action of PRL. Divalent (Fab)2 fragments, but not monovalent Fab, were also effective. These observations suggest that antibodies to the receptor, or the hormone itself, initiate a biological response possibly by cross-linking PRL receptors on the cell membrane, and that the entry of the PRL molecule, or fragments of it, into the intracellular compartment is not necessary for the biological action of PRL.

Effect of iodination on human growth hormone and prolactin: characterized by bioassay, radioimmunoassay, radioreceptor assay, and electrophoresis.

Human GH (hGH) and PRL (hPRL) were iodinated using lactoperoxidase. The iodinated hormones were characterized by RIA, radioreceptor assay (RRA), and bioassay (BA) using the Nb2 Node lymphoma cell line. The proportion of tracer that could bind to rat liver membranes or rabbit antibodies was determined, and the distribution of iodinated hormones was examined using polyacrylamide gel electrophoresis. Excess antibody was capable of precipitating 87.9% of the radioactivity associated with the hGH tracer and 86.0% of the hPRL tracer. The maximal specific binding to a liver membrane preparation averaged 67.3% of the [125I]iodo-hGH radioactivity and 48.8% of the [125I]iodo-hPRL radioactivity. The respective BA and RRA activity estimates for [125I]iodo-hGH averaged 90% and 114% of the activity measured by the RIA. For [125I]iodo-hPRL, the values were 75% by BA and 68% by RRA. The bioactivity profiles of iodinated hGH and hPRL shifted anodally on polyacrylamide gel electrophoresis in comparison to the bioactivity distribution of the respective uniodinated hormones. Iodine incorporation rather than oxidation appeared to be responsible for the shift. After electrophoresis, all eluates which contained significant radioactivity were active in the BA and RIA. Furthermore, specific activities calculated from the bioactive hormone and radioactivity in each electrophoretic segment agreed well with the average specific activity estimated from the amount of iodine incorporated into the protein peak upon gel filtration. These data suggest that hGH and hPRL to a major degree retain biological integrity after iodination.

Biochemical response of lymphoma cells to mitogenic stimulation by prolactin.

A previous study showed that cultured Nb 2 node rat lymphoma cells stopped replicating when transferred to medium supplemented with horse serum instead of fetal calf serum; resumption of growth could be induced by the addition of prolactin or other lactogens. The present study shows that in the absence of prolactin cells accumulated early in the G1 phase from which, on stimulation by the hormone, they proceeded through the cell cycle in a synchronized fashion. Ornithine decarboxylase and S-adenosyl methionine decarboxylase activities were closely related to the proliferative status of the cells. In stationary cultures the enzyme activity was barely detectable; following the addition of prolactin, the levels increased over 100-fold and displayed well-defined changes as the cells proceeded through the cell cycle. The results suggest the lymphoma cells are very useful for studying biochemical events resulting from the interaction of a mitogenic polypeptide hormone and its target cell.

A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum.

The replication of Nb 2 Node rat lymphoma cells in suspension culture is specifically stimulated by lactogenic hormones. Human (hPRL), ovine, bovine, and rat PRLs stimulated replication in a dose-dependent manner in the concentration range of 10 pg/ml to 1 ng/ml. Human, ovine, and bovine placental lactogens were similarly active. In addition, cell replication was stimulated by human GH (hGH), which is known to have lactogenic activity. Other hormones and growth factors examined were inactive. The growth stimulatory effects of hPRL and hGH were completely inhibited when excess anti-hPRL and anti-hGH, respectively, were added to the medium. A bioassay based on the response of the Nb 2 Node lymphoma cells to lactogenic hormones has been developed. Human serum stimulated cell replication. The effect was completely abolished if excess antibodies to both hPRL and hGH were present. The stimulation obtained with a number of human serum samples correlated very well with the sum of the hPRL and hGH concentrations in the sera, as determined by RIA (r = 0.95; P < 0.001). The concentrations of either hPRL or hGH in human serum could be individually determined by specifically blocking the growth stimulatory effect of the other hormone by adding excess anti-hGH or anti-hPRl. The sensitivity of this bioassay for PRL and hGH in serum exceeds that of RIAs.

Prolactin-stimulated growth of cell cultures established from malignant Nb rat lymphomas.

A malignant Nb rat lymphoma which in vivo is stimulated by estrogens has been established in suspension culture. The cultured cells grew readily in Fischer's medium supplemented with fetal calf serum (10%) and 2-mercaptoethanol (10(-4) M). If horse serum was substituted for fetal calf serum, population growth ceased; i.e., cultures became "stationary." Such stationary cultures could be induced to resume active growth by the addition of a pituitary hormone, prolactin (ovine, rat); concentrations as low as 10 pg/ml had a detectable effect. In contrast, other pituitary hormones or estrogens had little or no effect. The evidence in this and an accompanying paper suggests that prolactin (or related substances) has a role in the growth of some cancers of lymphoid origin in rats.

Evidence in vivo and in vitro of a role for the pituitary in the growth of malignant lymphomas in Nb rats.

The growth of a transplantable malignant lymphoma in Nb rats, the Nb 2 node, was accelerated in rats bearing an estrogen pellet and, in particular, in rats bearing transplanted tumors of the anterior pituitary gland. "Lymphoma cell growth-promoting activity" in the sera from the rats was detected and assayed using cultures of Nb 2 node cells. The "activity" of the sera paralleled the degree to which lymphoma growth was accelerated in the animals. Serum from normal rats at a concentration of 1% was moderately active in stimulating culture growth; serum from estrogenized rats was at least 10 times more active; serum from rats bearing pituitary tumors was extremely active and stimulated growth even at a concentration of 0.001% (1:10(5) dilution). In contrast, serum from hypophysectomized rats was devoid of activity, even if the animals were estrogenized. The evidence indicates (a) that the growth of the lymphoma in Nb rats was stimulated by factor(s) in the peripheral blood, the levels of which were subject to control by the pituitary, and (b) that estrogen stimulated lymphoma growth indirectly, through the pituitary gland.

The distribution of [3H]vinblastine in tumor and host tissues of Nb rats bearting a transplantable lymphoma which is highly sensitive to the alkaloid.

A new transplantable lymphoma in Nb rats responded dramatically to treatment with vinblastine (VLB). A single i.p. injection of VLB, 0.8 mg/kg, caused even highly advanced tumors to regress until they were no longer palpable. or investigation of the hypothesis that the oncolytic response may reflect a special affinity of VLB for the tumor, lymphoma-bearing rats were given an i.p. injection of -e13H]VLB, and the levels of radioactivity and [3H]VLB in the tumor and host tissues were determined as a function of time. Radioactivity was concentrated by the lymphoma relative to the blood (mostly as unchanged [3H]VLB) at levels that showed only a modest decline over a period of at least 48 hr. During this time the [2H]VLB in both the plasma and whit blood cell fraction of the blood declined markedly and continuously to very low levels. Thymus and lymph nodes resembled the lymphoma in showing a long-term retention of radioactivity. The levels of radioactivity in the spleen, liver, and bone marrow were initially much higher than that in the tumor but decreased markedly with time. In addition very little of the radioactivity remaining in the spleen and liver at 48 hr was due to [3H]VLB, and by this time the VLB concentration in these tissues was much lower than in the tumor. It is suggested that the chemotherapeutic response of the lymphoma may be related to the continuing presence of a significant concentration of VLB in this tumor after the plasma VLB had fallen to very low (subantimitotic) levels.