Labeling of murine mastocytoma cells in vitro with plasma tritiated thymidine-labeled animals.

40 min after injecting tritiated thymidine into an animal, 20-30% of the total plasma radioactivity is nonvolatile. This fraction decreases to about 6% 10 hr after the injection and 3% 24 hr after the injection. There appears to be material in this nonvolatile fraction that can label mastocytoma cells in culture. The labeling indices decrease with time after injection in the same way as the nonvolatile fraction. The 40 min plasma sample contains sufficient material to allow accurate assessment of the fraction of cells in S in culture after a 6 wk exposure. The circulating material is not apparently available for incorporation into those cells in cycle in the donor animal. The material appears to be related to the G(0) cell-specific pool that has been described elsewhere. The trichloroacetic acid-soluble or ethanol-soluble nonvolatile activity appears to contain thymine, and some thymidine-phosphorylated compounds.

Determination of rates of DNA synthesis in cultured mammalian cell populations.

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 microM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.

Formation of mast cell granules in cell cycle mutants of an undifferentiated mastocytoma line: evidence for two different states of reversible proliferative quiescence.

A heat-sensitive (hs, arrested at 39.5 degrees C, multiplying at 33 degrees C) and a cold-sensitive (cs, arrested at 33 degrees C, multiplying at 39.5 degrees C) cell cycle variant were isolated from an undifferentiated P-815 murine mastocytoma line. At the respective nonpermissive temperature, both the hs and the cs variant cells were reversibly arrested with a DNA content, typical of G1 phase. The cells of two cs variant subclones, when exposed to the nonpermissive temperature of 33 degrees C, formed metachromatically staining granules with an ultrastructure resembling that of mature mast cells. In addition, the cellular 5-hydroxytryptamine content underwent a marked increase, and the cells responded to compound 48/80 by degranulation as described for normal mast cells. On the other hand, in cells of two hs variant subclones, essentially no mast cell granules were detectable at either 33 or 39.5 degrees C. As previously reported, the cs cell cycle variant phenotype is expressed dominantly in heterokaryons obtained by fusing cs with wild-type cells, whereas hs cell cycle variant cells, similar to other hs mutants, were found to behave recessively under these conditions. Thus the state of proliferative quiescence induced in the cs cells at 33 degrees C is qualitatively different from the state of cell cycle arrest observed in hs cells at 39.5 degrees C and may represent a model for proliferative quiescence of differentiated cells in the intact organism.

Cholecystokinin(CCK)-A and CCK-B/gastrin receptors in human tumors.

Cholecystokinin (CCK)-A and CCK-B/gastrin receptors were evaluated with in vitro receptor autoradiography in 406 human tumors of various origins using a sulfated 125I-labeled CCK decapeptide analogue 125I-(D-Tyr-Gly, Nle28,3l)-CCK 26-33 and 125I-labeled Leu15-gastrin as radioligands. CCK-B/gastrin receptors were found frequently in medullary thyroid carcinomas (92%), in small cell lung cancers (57%), in astrocytomas (65%), and in stromal ovarian cancers (100%). They were found occasionally in gastroenteropancreatic tumors, breast, endometrial, and ovarian adenocarcinomas. They were either not expressed or rarely expressed in colorectal cancers, differentiated thyroid cancers, non-small cell lung cancers, meningiomas, neuroblastomas, schwannomas, glioblastomas, lymphomas, renal cell cancers, prostate carcinomas, and the remaining neuroendocrine tumors (i.e., pituitary adenomas, pheochromocytomas, paragangliomas, and parathyroid adenomas). CCK-A receptors were expressed rarely in tumors except in gastroenteropancreatic tumors (38%), meningiomas (30%), and some neuroblastomas (19%). The identified CCK-A and CCK-B receptors were specific and of high affinity in the subnanomolar range. The rank order of potency of various CCK analogues was: sulfated CCK-8 = L-364,718 >> nonsulfated CCK-8 = L-365,260 > or = gastrin for CCK-A receptors and sulfated CCK-8 > gastrin = nonsulfated CCK-8 > L-365,260 > L-364,718 for CCK-B receptors. CCK-B receptors could also be selectively and specifically labeled with a newly designed nonsulfated 125I-(D-Tyr-Gly, Nle28,31)-CCK 26-33. Gastrin mRNA measured by in situ hybridization was present in most CCK-B receptor-positive small cell lung cancers, breast tumors, and ovarian tumors, representing the molecular basis of a possible autocrine growth regulation of these tumors. Gastrin and CCK mRNAs were lacking in medullary thyroid cancers. Thus, these results may have pathogenic, diagnostic, differential diagnostic, and therapeutic implications.

Expression and localization of somatostatin receptor SSTR1, SSTR2, and SSTR3 messenger RNAs in primary human tumors using in situ hybridization.

Somatostatin receptor gene expression of SSTR1, SSTR2, and SSTR3 subtypes was evaluated by in situ hybridization in 55 human primary tumors shown to contain a high density of somatostatin receptors in binding assays. All 55 tumors expressed at least one SSTR subtype. Of 55 somatostatin receptor-positive tumors, 46 had SSTR2 mRNA; all 46 were characterized as having receptors with a high affinity for the synthetic analogue octreotide. Of 55 tumors, 12 expressed SSTR1, and 14 expressed SSTR3 mRNA. The subtype SSTR1 was expressed alone in 4 cases, SSTR2 was expressed alone in 33 cases, and SSTR3 was expressed alone in one case. In 4 cases, all 3 SSTR were expressed simultaneously. The cases having SSTR1 mRNA were identified in binding experiments with 125I-labeled somatostatin-14 and -28 analogues rather than with 125I-[Tyr3]-octreotide. Whereas meningiomas, neuroblastomas, pituitary adenomas, small cell lung carcinomas, lymphomas, and breast tumors expressed primarily a high abundance of SSTR2, carcinoids, islet cell carcinomas, medullary thyroid carcinomas, and ovarian tumors had a mixed distribution of the somatostatin receptor subtypes. This is the first demonstration of the presence of SSTR1, SSTR2, and SSTR3 in primary human tumors using in situ hybridization. Since these somatostatin receptor subtypes probably mediate distinct somatostatin actions, it may be worthwhile to search for subtype-specific analogues to use for the treatment and diagnosis of these tumors.

Y(1)-mediated effect of neuropeptide Y in cancer: breast carcinomas as targets.

Overexpression of selected peptide receptors in human tumors has been shown to represent clinically relevant targets for cancer diagnosis and therapy. Neuropeptide Y (NPY) is a peptide neurotransmitter mediating feeding behavior and vasoconstriction. It has never been shown to be involved in human cancer. We show here, using in vitro receptor autoradiography, a NPY receptor incidence of 85% in primary human breast carcinomas (n = 95) and of 100% in lymph node metastases of receptor-positive primaries (n = 27), predominantly as Y(1) subtype, whereas non-neoplastic human breast expressed Y(2) preferentially. Y(1) mRNA was detected in Y(1)-expressing tumors by in situ hybridization, whereas Y(2) mRNA was found in normal breast tissue. The strong predominance of Y(1) in breast carcinomas compared with Y(2) in normal breast suggests that neoplastic transformation can switch the NPY receptor expression from Y(2) to Y(1) subtype. Moreover, in Y(1)-expressing human SK-N-MC tumor cells, an NPY-induced, dose-dependent inhibition of tumor cell growth of >40% was observed, suggesting a functional role of NPY in cancer, mediated by Y(1). NPY should therefore be added to the list of small regulatory peptides related to cancer. The high incidence of Y(1) in in situ, invasive, and metastatic breast cancers allows for the possibility to target them for diagnosis and therapy with NPY analogues.

Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle.

At 0 degrees C, CHO cells efficiently incorporated [3H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37 degrees C, 15-25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0 degrees C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37 degrees C, but not at 0 degrees C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0 degrees C and reincubated at 37 degrees C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.

Control of ribonucleotide reductase in heat- and cold-sensitive mammalian cell-cycle mutants.

Two heat-sensitive (reversibly arrested in G1 phase at 39.5 degrees C, multiplying at 33 degrees C) and two cold-sensitive (reversibly arrested in G1 phase at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were tested for ribonucleotide reductase activity, using cells made permeable to nucleotides. After transfer of the heat-sensitive mutant cells to 39.5 degrees C, ribonucleotide reductase activity, similar to thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), but unlike DNA polymerase alpha (Schneider, E., Müller, B. and Schindler, R. (1985) Biochim. Biophys. Acta 825, 375-383), decreased rapidly and in parallel with numbers of cells in S phase, whereas in the cold-sensitive mutant cells brought to 33 degrees C, ribonucleotide reductase activity decreased approx. 8 h later than numbers of DNA-synthesizing cells. When arrested heat- or cold-sensitive mutant cells were returned to the permissive temperature, ribonucleotide reductase activities, similar to DNA polymerase alpha and to thymidine kinase in heat-sensitive mutants, increased essentially in parallel with reentry of cells into S phase, whereas the increase in thymidine kinase activity in the cold-sensitive mutants was previously shown to occur approx. one cell-cycle time later. This indicates that ribonucleotide reductase and thymidine kinase are coordinately expressed in the heat-sensitive, but independently regulated in the cold-sensitive mutants.

High gastrin and cholecystokinin (CCK) gene expression in human neuronal, renal, and myogenic stem cell tumors: comparison with CCK-A and CCK-B receptor contents.

Gastrin and cholecystokinin (CCK) are two major regulatory peptides synthesized by human gut and brain tissues as well as by selected tumors, in particular gastrin-producing neuroendocrine tumors. In the present study we have evaluated gastrin and CCK gene expression in a group of primary human tumors, including neuronal, renal, and myogenic stem cell tumors, using in situ hybridization techniques. In addition, CCK-A and CCK-B receptors were evaluated in the same group of tumors with receptor autoradiography. Most tumors had gastrin messenger ribonucleic acid (mRNA): 10 of 11 medulloblastomas, 5 of 5 central primitive neuroectodermal tumors, 11 of 11 Ewing sarcomas, 8 of 10 neuroblastomas, 4 of 4 Wilms' tumors, 5 of 5 rhabdomyosarcomas, and 10 of 10 leiomyosarcomas. CCK mRNA was restricted predominantly to Ewing sarcomas (9 of 11) and leiomyosarcomas (5 of 10). CCK-A and CCK-B receptors were not frequently found in these tumors, except for leiomyosarcomas. These data suggest that gastrin and CCK may play a previously unrecognized role in this group of human stem cell tumors. If the increased gastrin mRNA indeed translates into increased gastrin production, measurement of gastrinemia may have a diagnostic significance in the early detection of these tumors. As these two hormones have been reported to act as potent growth factors, they may be of pathophysiological relevance for patients with such stem cell tumors.

Somatostatin receptors in human prostate and prostate cancer.

Benign as well as malignant human prostatic tissues were evaluated for their content of somatostatin (SRIH) receptors (SRIH-R). In vitro receptor autoradiography techniques on cryostat sections were performed using 125I-labeled [Tyr3]octreotide as well as 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28 as radioligands. SRIH-R were identified in all normal and hyperplastic prostates in the smooth muscles of the stroma, whereas the glands did not express the receptors. Muscular nodules were strongly receptor positive as well. The receptors were of high affinity (Kd = 0.4 nmol/L) and high specificity for biologically active SRIH analogs; high affinity for SRIH-14, SRIH-28, and octreotide was detected, suggesting the presence of the SSTR2 receptor subtype. In situ hybridization studies confirmed the presence of SSTR2 messenger ribonucleic acid in these tissues. Primary prostate cancers did not have SRIH-R identified with 125I-labeled [Tyr3]octreotide. However, they were expressing SRIH-R identified with 125I-labeled [Leu8,D-Trp22,Tyr25]SRIH-28, with a high affinity for SRIH-14 and SRIH-28, but low affinity for octreotide. The receptors were located on tumoral cells. In situ hybridization studies revealed a preferential expression of SSTR1. Primary human prostate cancers, therefore, express a different SRIH-R subtype than benign prostate tissue. Several veins and the ganglion cells from the prostatic plexus in the surroundings of the tumors were expressing SRIH-R with high affinity for octreotide as well. These data suggest that the human prostate as well as prostate cancers may be targets for SRIH therapy; however, SRIH analogs with different selectivities for SRIH-R subtypes are required in each case.

Potent somatostatin undecapeptide agonists selective for somatostatin receptor 1 (sst1).

A family of analogues of des-AA(1,2,5)-[DTrp(8)/D2Nal(8)]-SRIF that contain a 4-(N-isopropyl)-aminomethylphenylalanine (IAmp) at position 9 was identified that has high affinity and selectivity for human somatostatin receptor subtype 1 (sst1). The binding affinities of des-AA(1,2,5)-[DTrp(8),IAmp(9)]-SRIF (c[H-Cys-Lys-Phe-Phe-DTrp-IAmp-Thr-Phe-Thr-Ser-Cys-OH], CH-275) (7), des-AA(1,5)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (CH-288) (16), des-AA(1,2,5)-[Tyr(7),DTrp(8),IAmp(9)]-SRIF (23), and des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-SRIF (25) are about (1)/(7), (1)/(4), (1)/(125), and (1)/(4) that of SRIF-28 (1) to sst1, respectively, about (1)/(65), (1)/(130), <(1)/(1000), and <(1)/(150) that of 1 to sst3, respectively, and about or less than (1)/(1000) that of 1 to the other three human SRIF receptor subtypes. A substitution of DTrp(8) by D2Nal(8) in 7 to yield des-AA(1,2,5)-[D2Nal(8),IAmp(9)]-SRIF (13) and in 16 to yield des-AA(1,5)-[Tyr(2),D2Nal(8),IAmp(9)]-SRIF (17) was intended to increase chemical stability, selectivity, and affinity and resulted in two analogues that were less potent or equipotent with similar selectivity, respectively. Carbamoylation of the N-terminus as in des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) increased affinity slightly as well as improved selectivity. Monoiodination of 25 to yield 26 and of 27 to yield 28 resulted in an additional 4-fold increase in affinity at sst1. Desamination of the N-terminus of 17 to yield 18, on the other hand, resulted in significant loss of affinity. Attempts at reducing the size of the ring with maintenance of selectivity failed in that des-AA(1,4,5,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (33) and des-AA(1,4,5,6,12,13)-[Tyr(2),DTrp(8),IAmp(9)]-SRIF (34) progressively lost affinity for all receptors. Both des-AA(1,2,5)-[DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (27) and des-AA(1,2,5)-[DCys(3),DTrp(8),IAmp(9),Tyr(11)]-Cbm-SRIF (29) show agonistic activity in a cAMP assay; therefore, the structural basis for the agonist property of this family of analogues is not contingent upon the chirality of the Cys residue at position 3 as shown to be the case in 18-membered ring SRIF octapeptides. None of the high affinity structures described here showed receptor antagonism. We have prepared the radiolabeled des-AA(1,2,5)-[DTrp(8),IAmp(9),(125)ITyr(11)]-SRIF ((125)I-25) and des-AA(1,2,5)-[DTrp(8),IAmp(9), (125)ITyr(11)]-Cbm-SRIF ((125)I-27), used them as in vitro tracers, and found them to be superior to des-AA(1,5)-[(125)ITyr(2),DTrp(8),IAmp(9)]-SRIF ((125)I-16) for the detection of sst1 tumors in receptor autoradiography studies.

Expression of somatostatin receptors in normal, inflamed, and neoplastic human gastrointestinal tissues.

The multiple actions of somatostatin are mediated by specific membrane-bound receptors present in all somatostatin target tissues, such as brain, pituitary, pancreas, gastrointestinal tract, and kidney. For instance, in the human gastrointestinal tract, three different types of tissue compartments express somatostatin receptors: the gastrointestinal mucosa, the peripheral nervous system, and the gut-associated lymphoid tissue, where the receptors are preferentially located in germinal centers. In all these cases, somatostatin binding is of high affinity and specific for bioactive somatostatin analogues. Somatostatin receptors are also expressed in pathological states, such as cancers. A particular abundance is found in neuroendocrine tumors of the gastrointestinal tract. Ninety percent of the carcinoids and a majority of islet cell carcinomas, including their metastases, usually have a high density of somatostatin receptors. Several different somatostatin-receptor subtypes can be expressed by these tumors, the SSTR2 subtype being the most frequently and abundantly expressed. The somatostatin receptors in tumors are identified with in vitro-binding methods, molecular biology techniques, or in vivo-imaging techniques; the latter allow the precise localization of the tumors and their metastases in the patients. Because somatostatin receptors in human gastroenteropancreatic tumors are functional, their identification can be used to predict the therapeutical efficacy of octreotide to inhibit excessive hormone release. Of differential diagnostic importance is the fact that other pathological processes in the gastrointestinal tract may be associated with a high density of somatostatin receptors. Ninety percent of lymphomas, including those with intestinal involvement express somatostatin receptors. Furthermore, a moderate number of colorectal carcinomas contain somatostatin receptors, whereas exocrine pancreatic carcinomas do not. Finally, an increased expression of SS receptors in nonneoplastic conditions, such as in intestinal veins in inflammatory bowel disease, has been recently observed. These observations demonstrate the ability of the human body to regulate SS receptors in a wide number of tissues and conditions.

Somatostatin receptors and their subtypes in human tumors and in peritumoral vessels.

Somatostatin receptors are expressed by a large variety of human tumors. In vitro receptor autoradiographic studies have shown that these tumors can express more than one somatostatin receptor subtype. Whereas the majority of tumors bind octreotide with high affinity, some, i.e., prostate tumors, bind octreotide with low affinity only. The discovery of five somatostatin receptor subtypes, sst1-5, by gene cloning has increased our understanding of somatostatin receptor structure and function. Using in situ hybridization techniques, we found that various human tumors, identified as somatostatin receptor-positive in binding studies, expressed sst2 mRNA in the majority of cases, whereas sst1 and sst3 were less frequent. Often, all three sst were expressed simultaneously. In another recent in situ hybridization study, primary prostate cancers were shown to preferentially express sst1, rather than sst2 or sst3. Moreover, a high incidence of sst5 was found in growth hormone (GH)-producing pituitary adenomas and, to a lesser extent, in active pituitary adenomas; gastroenteropancreatic (GEP) tumors showed all possible combinations, but with a predominance of sst2. Overall, the presence of sst2 mRNA and/or sst5 generally correlated with the presence of octreotide-binding sites, but with exceptions. These results indicate the highly variable abundance of sst mRNAs in individual somatostatin receptor-containing tumors. Somatostatin receptors were not only found in tumoural tissue, but also in the peritumoral vascular system. This was particularly well studied in colorectal carcinomas, where the peritumoral veins were shown to express in all cases a high density of somatostatin receptors, probably of the sst2 type, binding octreotide with high affinity. Therefore, the host peritumoral vascular system may be a possible target of somatostatin action in tumor development. Somatostatin may act locally on tumor growth through two different mechanisms dependent on local somatostatin receptor expression: through direct action on tumor cells or through action on peritumoral vessels, which may alter the hemodynamics of the tumoral blood circulation.

A selective analog for the somatostatin sst1-receptor subtype expressed by human tumors.

Somatostatin mediates its actions through five different somatostatin receptor subtypes, sst1-sst5. Recently, the somatostatin analogs des-AA1,2,5-[D-Trp8, IAmp9]somatostatin and des-AA1,5-[Tyr2, D-Trp8, IAmp9]somatostatin were synthesized and shown to be sst1-selective when tested in COS-7 cells transfected with each of the sst subtypes. In the present study, we tested the binding affinity and specificity of the iodinatable analog in primary human tumors expressing various sst subtypes, selected on the basis of in situ hybridization experiments. Des-AA1,5-[Tyr2, D-Trp8, IAmp9]somatostatin was found to have a high affinity, comparable to that of the natural somatostatin-28, for sst1-expressing tumors such as prostate cancers. However, it had no affinity for tumors expressing the sst2, sst3, or sst5 subtypes. For comparison, the somatostatin analogs octreotide or Tyr3-octreotide have no affinity for sst1-expressing tumors, but high affinity for sst2- and sst5-expressing tumors and intermediate affinity for sst3-expressing tumors. These data represent the first characterization of a sst1-selective analog in human tumors; it may be of potential use in the therapy of sst1-expressing tumors as an antiproliferative agent, as well as providing a lead compound for the development of more potent sst1-selective radioligands for in vivo tumor scintigraphy.

Determination of thymidine in serum used for cell culture media.

Thymidine concentrations in serum used for cell culture media were determined with an assay based on isotope dilution. In this assay, incorporation of (3H)-thymidine into DNA of cultured cells was measured in the presence of 5 and 20% serum as a function of the concentration of unlabeled thymidine added to the medium. In three batches of horse serum, thymidine concentrations were 0--0.17 micron, while in fetal calf serum values of 0.75--2.1 micron were obtained. Dialysis of serum resulted in a reduction of thymidine levels by factors of at least 10.

Somatostatin receptor subtypes sst1, sst2, sst3 and sst5 expression in human pituitary, gastroentero-pancreatic and mammary tumors: comparison of mRNA analysis with receptor autoradiography.

Using in situ hybridization techniques with selective oligoprobes, the gene expression of sst1, sst2, sst3 and sst5 was studied in a series of 32 human pituitary adenomas, 28 breast tumors and 21 endocrine gastroentero-pancreatic tumors, shown to express somatostatin receptors to variable extents. In most of these tumors the sst2 receptor subtype was abundantly expressed, even though a significant number of pituitary adenomas, breast and gastroentero-pancreatic tumors expressed sst1 and/or sst3 as well. A very high incidence of the sst5 subtype was found in growth hormone-producing pituitary adenomas and, to a lesser extent, in inactive pituitary adenomas, whereas breast tumors seldom expressed sst5; gastroentero-pancreatic tumors showed all possible combinations of sst expression, with, however, a predominance of sst2 and sst1. Overall, the presence of sst2 mRNA and/or sst5 mRNA generally correlated with the presence of octreotide binding sites. A lack of octreotide binding sites corresponded with a lack of sst2 mRNA. Several tumors exhibiting a low number of octreotide binding sites had no measurable sst2 mRNA, despite abundance of beta-actin mRNA, suggesting in these cases a very low abundance of sst mRNAs or a too low sensitivity of the in situ hybridization methodology. In all other cases, the method allowed precise localization of the respective mRNAs on the tumor tissue, notably in breast tumors with non-homogeneous receptor distribution. Tumors without measurable amounts of somatostatin receptors had no detectable sst mRNA. Our results indicate a highly variable abundance of the various sst mRNAs in individual somatostatin receptor-containing tumors.

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons.

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.