Primary Tissue Culture Cells Research Articles

Highlights of This Issue

Mechanistic studies of patient-derived tumor tissues are often limited by sample size and tumor cell content. Methods for culturing primary cells ex vivo over a feeder layer are suitable for certain applications, but lack utility in highly sensitive assays like single cell sequencing due to potential contamination by cells from the feeder layer. Here, Prasad and colleagues detail a novel method of propagating dissociated primary cells in tissue culture using defined growth medium with inhibitors of ROCK, TGFβ, and BMP signaling. This method supports the expansion of a variety of cell types, including stromal, luminal, basal, and stem cell subpopulations that are compatible with cell sorting applications and downstream -omics analysis. This method maximizes the utility of primary tissue samples, and broadens the horizon for ex vivo analyses.Mouse models of hepatocellular carcinoma (HCC) often fail to capture the intricacies of human disease, which may influence the clinical applicability of data derived from these models. Here, Friemel, Frick, and colleagues demonstrate the feasibility of using genomic, morphologic, and immunohistochemical analyses to stratify genetically engineered mouse models along eitiologic criteria, matching them with phenotypes observed in human HCC. The authors posit that this data will allow for better alignment of mouse models with HCC subtypes, thereby increasing their clinical relevance.Hypoxia-inducible factor (HIF)-1α is a key regulator of the cellular response to hypoxia. HIF-1α plays a major role in hypoxic tumor metabolism and owns the potential to be targeted for therapy. However, the mechanism by which cancer cells can adapt to HIF-1α deletion is poorly understood. Integrated metabolomic, proteomic, and transcriptomic analysis by Valli and colleagues has now revealed that HIF-1α-knockout colon cancer cells upregulate creatine metabolism and glucose transporter GLUT14 in response to hypoxic stress. Their data elucidate that colon cancer cell hypoxic metabolism can bypass HIF-1α deletion, provide new insight into the mechanisms underlying hypoxic stress responses, and highlight potential avenues for targeted therapy in colorectal cancer.The cell cycle inhibitor p21CIP1 has well-known roles in regulating cellular proliferation, but previous work has shown that it also exerts pro-metastatic effects in mammary tumor models via an unknown mechanism. Here, Benard and colleagues have defined the role of p21CIP1 in promoting cancer stem cell-like phenotypes, demonstrating that its expression is associated with increased Wnt/β-catenin pathway activity through TCF1 and Cyclin D1. Knockout of p21 was concordantly associated with reduction of cancer stem cell properties, inhibition of tumorsphere formation, and suppression of tumor-initiating potential in vivo. The data suggest that, despite its classical role in suppressing cell cycle progression, p21 is also a strong promoter of cancer stem cell properties in mammary cells.

Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

Abstract 5195: Novel patient-derived xenograft and primary tissue culture cells for preclinical study in sarcoma

Abstract Sarcomas are a group of malignant tumors displaying a wide diversity of pathological appearances and clinical responses due to the mesenchymal origin. Although certain sarcomas are characterized by the presence of unique fusion genes and mutations, the molecular backgrounds for tumorigenesis, metastasis, and resistance to treatments are remained to be further clarified in sarcomas. To improve clinical outcome, precise diagnosis followed by optimized therapy has long been desired in this field. Recently, clinical trials for application and re-localization of molecular targeting drugs have been conducted, and several drugs have been proven to effective in sarcomas. Preclinical models are indispensable to furthering our understanding of molecular background and facilitate novel therapeutic strategy. However, because of low prevalence the models are quite limited in sarcomas. To this end, we launched a project to establish patient-derived xenografts (PDXs) and primary culture cells of sarcomas. From April 2014 through October 2015, we implanted surgical tumor tissues from 76 sarcoma patients into highly immune-deficient NOG mice, and banked them after three passages. The identities of sarcomas were histologically confirmed by a certified pathologist, and the utility of PDXs were confirmed by re-growing them after storage in liquid nitrogen. 17 PDXs were prepared for preclinical study including myxofibrosarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor, liposarcoma, leiomyosarcoma, osteosarcoma and clear cell sarcoma. Tissue cultures were also conducted by utilizing surgical specimens or PDXs. 158 sarcoma tissue samples were subjected to primary culture. Five immortalized cell lines, and 33 primary cultured cell lines with a minimum number of passages were established. The former includes leiomyosarcoma and undifferentiated pleomorphic sarcoma, and the latter includes additional 10 subtypes such as myxofibrosarcoma, malignant peripheral nerve sheath tumor, liposarcoma, osteosarcoma, clear cell sarcoma, etc. Moreover, the molecular backgrounds of the established sarcoma PDXs and tissue cultured cells were evaluated by targeted sequencing using an original cancer gene panel (NCC oncopanel), which can detect targetable mutations and gene fusions. Clinical and pathological observations of the donor patients will be linked to the characters of PDXs and tissue cultures. The applications of these in vivo and in vitro models for preclinical study will be our next challenge. Citation Format: Xiaoqing Pan, Mami Takahashi, Hitoshi Ichikawa, Akihiko Yoshida, Akira Kawai, Toshio Imai, Tadashi Kondo. Novel patient-derived xenograft and primary tissue culture cells for preclinical study in sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5195.

PAR‐2 receptor‐induced intracellular signalling in isolated intact human eccrine sweat gland cells and primary tissue culture cells

PAR‐2 receptors can evoke intracellular signalling and anion transport in a cell line derived from human sweat glands, however, it is unknown whether PAR‐2 receptors are present and play a role in intact human sweat gland secretory cells. PAR‐2 receptors were investigated in isolated sweat glands and tissue culture cells derived from primary explants. Skin samples were obtained by informed consent and ethics approval from volunteers for immunohistochemistry and sweat gland isolation for; Western blot analysis; calcium imaging or explanted for tissue culture. Immunohistochemistry and Western blot using an anti human PAR‐2 antibody demonstrated the presence of these receptors in sweat gland secretory cells. Calcium imaging experiments using trypsin and the agonist peptide SLIGKV showed that both could increase intracellular calcium levels in intact glands and in the primary sweat gland cultures. Results also demonstrated that the effects of the agonist peptide were reversible upon washout, but not trypsin, and that the reverse peptide VKGILS failed to initiate changes in intracellular calcium. The results of this study demonstrate that PAR‐2 receptors are present in isolated human sweat gland secretory cells and in primary cultured cells and that these receptors are functionally active and can induce increases in intracellular calcium.The study was supported by Glasgow Caledonian University.

Deregulated E2F Activity Induces Hyperplasia and Senescence-Like Features in the Mouse Pituitary Gland

The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumors, we generated transgenic mice expressing an inducible E2F3 protein in the intermediate lobe of the pituitary gland. We found that short-term deregulation of E2F activity, similar to the earliest stages of Rb loss, is able to induce abnormal proliferation of otherwise quiescent melanotrophs. However, while long-term exposure to deregulated E2F activity results in hyperplasia of the intermediate lobe, it did not lead to tumor formation. In fact, melanotrophs become insensitive to sustained E2F stimulation and enter an irreversible senescence-like state. Thus, although deregulated E2F activity results in hyperproliferation, it is not sufficient to mimic loss of Rb, sustain proliferation of melanotrophs, and ultimately induce pituitary tumors. Similarly, we found that primary cells in tissue culture become insensitive to sustained E2F3 activation and undergo premature senescence in a pRB-, p16Ink4a-, and p19Arf-dependent manner. Thus, we conclude that deregulated E2F activity is not sufficient to fully mimic loss of Rb due to the engagement of a senescence response.

Prognostic significance of aneuploidy in non-small cell lung cancer (NSCLC) patients (pts)

7319 Background: DNA content abnormalities assessed with flow cytometry are linked with a poor prognosis in many cancer types, including NSCLC. The aim of this study was to determine prognostic significance of aneuploidy assessed with conventional cytogenetic analysis in NSCLC. Methods: Study group included 89 consecutive NSCLC pts (15 females and 74 males, mean age of 61 years) who underewent pulmonary resection between April 1997 and July 1999. Pathologic stage distribution was as follows: I (27 pts), II (23 pts), III (36 pts) and IV (3 pts). 54 pts had sqaumous cell carcinoma, 22 -adenocarcinoma, 6 –large cell carcinoma and 7 –mixed NSCLC. Tumor ploidy was determined by karyotyping of primary tissue culture cells derived from postoperative specimens. G-banding with Wright stain and further analysis was performed according to recommendations of the International System of Human Cytogenetics Nomenclature. Results: Aneuplody was present in 33 tumors (37%), more frequently in adenocarcinoma (25 pts, 71%) than in squamous cell carcinoma (8 pts, 15%, p<0.001). No correlation of aneuploidy with grade (p=0.70), stage (p=0.29), age (p=0.69) and sex (p=1.00) was found. Median survival of pts with and without aneuploidy was 15 and 38 months, respectively (log rank; p=0.028). In the multivariate analysis, aneuploidy was the strongest adverse factor for survival (HR=1.92, 95% CI 1.11–3.32; p=0.020), followed by tumor stage (HR=1.59, 95% CI 0.92–2.74; p=0.094). Conclusion: NSCLC aneuploidy, as evaluated by conventional cytogenetic analysis, occurs more frequently in adenocarcinoma than in squamous cell carcinoma, and is an independent predictor of poor prognosis. No significant financial relationships to disclose.

Gene expression phenotypic models that predict the activity of oncogenic pathways.

High-density DNA microarrays measure expression of large numbers of genes in one assay. The ability to find underlying structure in complex gene expression data sets and rigorously test association of that structure with biological conditions is essential to developing multi-faceted views of the gene activity that defines cellular phenotype. We sought to connect features of gene expression data with biological hypotheses by integrating 'metagene' patterns from DNA microarray experiments in the characterization and prediction of oncogenic phenotypes. We applied these techniques to the analysis of regulatory pathways controlled by the genes HRAS (Harvey rat sarcoma viral oncogene homolog), MYC (myelocytomatosis viral oncogene homolog) and E2F1, E2F2 and E2F3 (encoding E2F transcription factors 1, 2 and 3, respectively). The phenotypic models accurately predict the activity of these pathways in the context of normal cell proliferation. Moreover, the metagene models trained with gene expression patterns evoked by ectopic production of Myc or Ras proteins in primary tissue culture cells properly predict the activity of in vivo tumor models that result from deregulation of the MYC or HRAS pathways. We conclude that these gene expression phenotypes have the potential to characterize the complex genetic alterations that typify the neoplastic state, whether in vitro or in vivo, in a way that truly reflects the complexity of the regulatory pathways that are affected.

Anti-adhesive activity of sulphated exopolysaccharides of microalgae on attachment of red sore disease-associated bacteria and helicobacter pylori to tissue culture cells.

Because of the affinity of certain bacterial species for sulphated glycoconjugates exposed on the epithelial cells of susceptible hosts, we hypothesized that sulphated exopolysaccharides of microalgae can be used in anti-adhesive therapies against bacterial infections, both in cold- and warm-blooded animals. In this study we found that adhesion of the human pathogen Helicobacter pylori to the HeLa S3 cell line, and adhesion of the fish pathogens Vibrio campbellii, V. ordalii, Streptococcus saprophyticus, and Aeromonas veronii to spotted sand bass primary tissue culture cells, can be effectively blocked with the various sulphated exopolysaccharides used.

LDL oxidation and human placental trophoblast and macrophage cytotoxicity.

We have previously shown that LDL is oxidized by placental cells in primary tissue culture and that this process causes cytotoxicity proportional to LDL oxidation in the presence of sex steroid hormones. Here we describe further experiments linking LDL oxidation to placental cell cytotoxicity. Trophoblast and macrophages were isolated from healthy elective caesarean section placentas by enzymatic digestion and separated by centrifugation on a 40% Percoll gradient and maintained in primary culture for up to 5 days. LDL was oxidized by exposure to 5 microM CuCl2, cells were incubated in the absence of albumen to favor oxidation, and cytotoxicity was measured by 51Cr release from prelabelled cells and cell protein content. Native LDL was oxidized by both cell types with a 10%-50% increase in lipid peroxides and an approximately 4-fold increase in TBARS formation. Increasing concentrations of native LDL and oxidized-LDL increased 51Cr release and diminished protein content in cells incubated in HAM's F-10 medium. Addition of 5 microM Cu2+ augmented cytotoxicity of LDL in macrophage and trophoblast culture, but more in macrophages than trophoblast. Cytotoxicity was diminished by adding 0.001-0.1 mM EDTA to the system, diminishing 51Cr release from 91 +/- 0.5 to 40.8 +/- 1.0% in macrophages and 54.2 +/- 1.2 to 33.1 +/- 1.3% in trophoblast (P < 0.001 in both instances). Similarly, the absence of transition metal ion in culture (MEM medium) blocked an increase in 51Cr release compared to its presence (HAM's F-10 medium). An antioxidant, butylated hydroxytoluene, diminished 51Cr release and LDL electrophoretic mobility in HAM's F-10 medium in placental macrophage culture. LDL oxidation injures placental macrophages and trophoblast, the former more than the latter. The process is LDL- and transition metal ion-dependent and is inhibited by antioxidant. This model of LDL oxidation and placental cell damage in vitro provides a basis for studying mechanisms of placental dysfunction and senescence in human pregnancy.

The nef gene products of both simian and human immunodeficiency viruses enhance virus infectivity and are functionally interchangeable.

Adult rhesus macaques infected with nef-defective simian immunodeficiency virus (SIV) exhibit extremely low levels of steady-state virus replication, do not succumb to immunodeficiency disease, and are protected from experimental challenge with pathogenic isolates of SIV. Similarly, rare humans found to be infected with nef-defective human immunodeficiency virus type 1 (HIV-1) variants display exceptionally low viral burdens and do not show evidence of disease progression after many years of infection. HIV-1 Nef induces the rapid endocytosis and lysosomal degradation of cell surface CD4 and enhances virus infectivity in primary human T cells and macrophages. Although expression of SIV Nef also leads to down-modulation of cell surface CD4 levels, no evidence for SIV Nef-induced enhancement of virus infectivity was observed in earlier studies. Thus, it remains unclear whether fundamental differences exist between the activities of HIV-1 and SIV Nef. To establish more clearly whether the SIV and HIV-1 nef gene products are functionally analogous, we compared the replication kinetics and infectivity of variants of SIVmac239 that either do (SIVnef+) or do not (SIV delta nef) encode intact nef gene products. SIVnef+ replicates more rapidly than nef-defective viruses in both human and rhesus peripheral blood mononuclear cells (PBMCs). As previously described for HIV-1 Nef, SIV Nef also enhances virus infectivity within each cycle of virus replication. As a strategy for evaluating the in vivo contribution of HIV-1 nef alleles and long terminal repeat regulatory sequences to the pathogenesis of immunodeficiency disease, we constructed SIV-HIV chimeras in which the nef coding and U3 regulatory regions of SIVmac239 were replaced by the corresponding regions from HIV-1/R73 (SIVR7nef+). SIVR7nef+ displays enhanced infectivity and accelerated replication kinetics in primary human and rhesus PBMC infections compared to its nef-defective counterpart. Converse chimeras, containing SIV Nef in an HIV-1 background (R7SIVnef+) also exhibit greater infectivity than matched nef-defective viruses (R7SIV delta nef). These data indicate that SIV Nef, like that of HIV-1, does enhance virus replication in primary cells in tissue culture and that HIV-1 and SIV Nef are functionally interchangeable in the context of both HIV-1 and SIV.

Localization of foot and mouth disease virus RNA in tissue culture infected cells via in situ polymerase chain reaction.

Foot and mouth disease virus RNA was visualized in infected primary tissue culture cells by in situ PCR incorporating digoxigenin-labeled dUTP. The viral RNA polymerase gene was used as a target for amplification. Infected cells revealed cytoplasmic staining, predominantly perinuclear. The intensity of staining was in proportion to the degree of cytopathology observed and similar to the results obtained using immunoperoxidase staining. The in situ PCR technique for FMDV detection could be applied to formalin-fixed samples and be useful for the study of persistent infections.

Characteristics of glutamine transport in primary tissue culture of rat skeletal muscle

Glutamine transport was studied in preconfluent monolayered, mononucleated myoblasts (4 days old) and in fused, multinucleated, differentiated myotubes (10 days old), both prepared from neonatal rat skeletal muscle. The initial (60 s) rate of 50 microM glutamine uptake in myoblasts and myotubes was stereospecific, saturable, and largely (80%) Na+ dependent. At glutamine concentrations of 0.01-1 mM, Na(+)-dependent uptake showed saturation kinetics: in myoblasts, the Michaelis constant (Km) was 197 +/- 38 microM, maximum velocity (Vmax) was 1,165 +/- 60 pmol.min-1.mg protein-1; in myotubes, Km was 174 +/- 51 microM and Vmax was 1,435 +/- 47 pmol.min-1.mg protein-1. The Na(+)-dependent glutamine uptake was Li+ tolerant in both myoblasts and myotubes. The Na(+)-dependent uptake of 50 microM L-[3H]glutamine was investigated in the presence of various amino acids at 0.01-10 mM. Histidine and asparagine competitively inhibited glutamine uptake, but inhibition by serine was noncompetitive; glutamate, arginine, leucine, and 2-aminobicyclo(2,2,1)heptane-2-carboxylate (BCH) had no significant inhibitory effects; 2-(methyl-amino)isobutyrate (MeAIB) caused a small but significant inhibition. In parallel with a stimulation of glucose transport, addition of insulin stimulated Na(+)-dependent glutamine uptake within 1 h by a maximum of 27% in myoblasts and 42% in myotubes (half-maximal stimulation at 0.3 nM insulin). Glucagon had no effect. Kinetic analysis revealed that the insulin-stimulated increase in glutamine transport was due to a Vmax effect, which was cycloheximide inhibitable. The insulin-stimulated increase was Li+ tolerant and not inhibited by MeAIB or cysteine at 1 mM. The results indicate that the predominant glutamine transporter of neonatal rat skeletal muscle cells in primary tissue culture in System Nm. System Nm also appears to be the major insulin-sensitive glutamine transport component in skeletal muscle. Primary muscle culture appears to be a useful preparation for studying glutamine transport and its regulation.

Calcium is released by exocytosis together with catecholamines from bovine adrenal medullary cells.

We have tested the hypothesis that exocytosis is a possible export route for calcium from bovine adrenal medullary cells. After prelabelling cells in primary tissue culture with 45Ca, evoked 45Ca export and catecholamine secretion show the same time course, a similar fraction of the total pool of 45Ca and catecholamine is released, and the same concentrations of carbamylcholine or KCl are required for half-maximal triggered release. Increasing the osmolarity of the extracellular medium or treating the cells with botulinum toxin type D inhibits both evoked catecholamine secretion and 45Ca export to the same extent without inhibiting 45Ca influx. Incorporation of 45Ca into chromaffin granules is very slow, however, and incorporated 45Ca is not immediately releasable. 45Ca entering the cell during short-term stimulation is not found in the releasable pool during a second period of triggered secretion. Our data suggest that chromaffin granules are the largest pool of intracellular calcium in bovine adrenal medullary cells and that most of the calcium in chromaffin granules does not rapidly exchange with cytoplasmic Ca, but can be released directly by exocytosis. Exocytosis does not appear to play a major role in exporting Ca that enters the cell during short-term stimulation.

Electrophysiological properties of glial cells: comparison of brain slices with primary cultures

Intracellular recordings were obtained from glial cells in the CA1 region of rat hippocampal slices to compare their electrophysiological properties with the previously reported properties of glial cells in primary tissue culture. The average resting potential was −77 mV and the average input resistance was 3.2 MΩ. Barium (10 mM) depolarized glial cells in brain slices and increased input resistance, but barium action potentials which have been observed in primary cultures, were not observed in brain slices. γ-aminobutyric acid (GABA) and glutamate depolarized glial cells. Spontaneous oscillations of membrane potential were observed occasionally. The mechanism underlying these responses are unknown as yet.

Fractionation of two mosquitocidal activities from alkali-solubilized extracts of Bacillus thuringiensis subspecies israelensis spores and parasporal inclusions

Incubation of Bacillus thuringiensis subsp. israelensis spore-crystal mixtures with alkaline solutions gave a soluble fraction that was toxic to both Aedes aegypti and Drosophila melanogaster larvae, as well as being cytolytic to both primary tissue culture cells derived from D. melanogaster embryos and sheep red blood cells. Sephadex G 75 gel filtration chromatography of the alkali-solubilized material resolved two fractions. The first fraction to elute from the columns was mosquitocidal but not cytolytic. The second fraction was cytolytic as well as mosquitocidal. In addition, only the cytolytic fraction had larvicidal activity against D. melanogaster. A protein, of approximately 27,000 Da having cytolytic activity, mosquitocidal activity, and toxic to D. melanogaster larvae, was purified from the second fraction. The rate of in vitro alkali solubilization of the mosquitocidal-noncytolytic activity was different from that of the mosquitocidal-cytolytic activity.

N-myc can cooperate with ras to transform normal cells in culture.

N-myc, a cellular gene bearing homology to the c-myc protooncogene, is frequently amplified and overexpressed in a highly restricted set of related tumors, most notably neuroblastomas and retinoblastomas. We have examined the possibility that N-myc may play a causal role in the genesis of these tumors by defining its ability to transform primary cells in tissue culture. Using an N-myc expression construct capable of producing constitutively deregulated levels of full-length murine N-myc mRNA, we demonstrate that a deregulated N-myc gene can cooperate with the activated Ha-ras oncogene to cause tumorigenic conversion of normal embryonic fibroblasts in a manner indistinguishable from the deregulated c-myc oncogene. Cell lines established from N-myc/ras-transformed foci express high levels of the N-myc gene, and such lines are similar to c-myc/ras transformants in their ability to grow in soft agar and cause tumors in syngeneic rats. These results illustrate that N-myc does encode a c-myc-like transforming activity and that this transforming activity is not specific for the very restricted set of tumors in which N-myc is normally amplified or overexpressed.

Membrane potential and Ca influx in hypoxic and normoxic carotid body type-I cells.

The carotid body is described as being a chemoreceptor since 1928 (de Castro). Under hypoxia, the natural stimulus, transmitter like dopamine, and Ach are released (Fidone et al., 1982; Eyzaguirre et al., 1965) and chemoreceptor discharge is increased (Delpiano and Acker, 1980). Till now it is not clear, whether this chemoreceptive process is located in the glomus cell membrane or in the adjacent nerve endings. Working with cells in primary tissue culture we can investigate the stimulation process on isolated carotid body cells. As previous studies showed (Pietruschka and Schafer, 1976) fine structure of these cells, especially the dense cored vesicles, is preserved in culture.

Resistance to ixodid tick infestation induced by administration of tick-tissue culture cells.

Primary tissue culture cells of developing larvae of Amblyomma americanum were administered to guinea-pigs never previously exposed to ixodid ticks. Guinea-pigs were given 1 X 10(6) primary culture cells on Days 0, 7 and 21 by subcutaneous injection and challenged with male and female A. americanum on Day 35. A significant degree of induced host tick resistance was expressed by reduced engorgement weight of females, reduced oviposition by those females which did obtain a blood meal, and by death of ticks at the attachment site. Resistance induced by A. americanum primary culture cells stimulated a significant degree of resistance to infestation with Dermacentor andersoni adults.

Primary tissue culture of human adrenocortical Conn's adenomata. Bromocriptine as a possible agonist-antagonist of angiotensin at the cellular level.

Angiotensin II-induced the hypertrophy of the cytoplasmic compartment and significantly increased (5-3H)uridine incorporation into RNA species by Conn's human adult adenomatous cells in primary tissue culture. On its own, bromocriptine, while enlarging only the nucleolar compartment, also intensely stimulated the incorporation of (5-3H)uridine into RNA species by the cultured adrenocortical adenomatous cells. However, an equimolar mixture of angiotensin II and bromocriptine was totally ineffective, eliciting no change in cellular morphometry or isotope incorporation with respect to the control specimens run in parallel. The present findings support the view that bromocriptine can influence the metabolism of Conn's cells directly at the cellular level by acting as an agonist-antagonist of angiotensin.

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E 3 has 12% the affinity of estradiol (E 2) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E 3(10 −10 M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E 3 to be only slightly less effective than E 2. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E 3 while E 2 is metabolized to estrone. Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.