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Cancer cell lines have been, and will continue to be, important tools in oncology research, but there are major limitations in the fidelity of existing cell lines as a model of human cancers. Therefore, the aim of our study was to generate and characterize patient-specific cell lines from solid tumors that more faithfully recapitulate the primary tumor genetic and morphologic characteristics. We performed cell line derivation on a primary human breast adenocarcinoma (ER+/PR+/HER2-), a lung adenocarcinoma, a high-grade serous ovarian carcinoma, and an endometrioid ovarian carcinoma procured from a commercial tissue bank. Portions were snap-frozen for molecular analysis, and the remainder was minced and processed for cell culture. Cells were cultured and the resulting monolayer was stained by immunocytochemistry with a pan-keratin antibody for the presence of epithelial cells. Any contaminating fibroblasts were removed. We extracted DNA from the resulting breast and lung cancer cell lines at low, medium, and high passage numbers (approximately 20, 40, and 150 population doublings), as well as the corresponding primary tumor specimen, and a 250K SNP array was performed. These data were combined with that of nine breast cancer and 13 lung cancer cell lines from the GEO database, and subjected to genotyping analysis using the BLRMM algorithm in the Affymetrix Genotyping Console. The percent identity between the primary tumor tissue and the cell line was calculated from the number of identical SNP calls divided by the total number of SNP calls. Probesets resulting in “no call” genotyping results due to poor quality reads for either sample were not included in the analysis. For comparison, unrelated cancer cell line data of the same tumor type was included in the analysis. The cell lines reported here have grown for a minimum of 25 population doublings without evidence of cell culture crisis. The lung cancer, serous ovarian, and endometrioid ovarian cancer cell lines were positive for pan-keratin staining by immunocytochemistry. Both ovarian cancer subtypes exhibited tumorsphere formation under non-adherent culture conditions, whereas the lung cancer cell line formed loose aggregates. The percent identity between the primary carcinoma and cell line at 20, 40, and 150 population doublings was determined using a SNP call quality threshold of 0.1, resulting in an average 100K and 56K highest quality reads for breast and lung. The percent identity for the breast cancer cell line resulted in 98.7%, 97.4%, and 98.3% (mean +/- SD = 98.2% +/- 0.7%). The percent identity for the lung adenocarcinoma cell line at 20, 40, and 150 population doublings was 89.5%, 84.4%, and 87.2% (mean +/- SD = 87.0% +/- 2.5%). In comparison, the average % identity between unrelated existing breast cancer cell lines was 61.0% (+/- 3.1%) and between unrelated lung cancer cell lines was 58.7% (+/- 3.7%). These patient-specific breast and lung cancer cell lines exhibit genetically stable, extended growth from a primary tissue specimen for a minimum of 150 population doublings. We continue to measure extended growth in the ovarian cancer cell lines as well as a colon adenocarcinoma. Novel cell lines may be derived using this technology to represent specific patient groups and to serve the needs of researchers in precision medicine performing drug screening, target discovery, and the development of companion diagnostics. Citation Format: Naghmeh Salimi, Jeffrey D. Kent, Alex Chao, Bryan E. Roberts, Agoston Agoston, Elin S. Agoston. Patient-specific cancer cell line derivation methodology results in genetic stability across 150 population doublings. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 33.

We investigated the ability of a novel polyphosphazene polyelectrolyte, poly[di(sodium carboxylatoethylphenoxy)phosphazene] (PCEP) to enhance antigen-specific immune responses. BALB/c mice were immunized once subcutaneously with either bovine serum albumin (BSA) or influenza virus X:31 antigen alone, or in combination with PCEP, or either of the adjuvants poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) and alum. Both PCEP and PCPP significantly enhanced serum antigen-specific total IgG, IgG1 and IgG2a antibody titers, and these responses were highest in PCEP-immunized mice. Alum induced only a modest enhancement of antibody responses. Reducing the dose of X:31 antigen by 25-fold had no effect on antibody responses in mice immunized with PCPP and PCEP, but resulted in reduced titers in those immunized with alum. Analysis of X:31 antigen-specific cytokines revealed that alum and PCPP were associated with a predominantly IL-4 response. In contrast, PCEP was associated with production of both IFNγ and IL-4. We conclude that PCEP is a potent enhancer of antigen-specific Th1 and Th2 immune responses and is a promising adjuvant for vaccine applications.

The advent of modern molecular biology has provided us with a means of producing antigens with unprecedented ease and precision. It is ironic that these new methodologies generate purified antigens that do not generally induce a strong immune response in the absence of an effective adjuvant. The development of improved vaccine adjuvants for use in humans has therefore become a priority area of research. Nevertheless, research on adjuvants has lagged seriously behind the work done on antigens. For decades the only adjuvant widely used in humans has been alum. Saponin and its purified component Quil A, complete Freund’s adjuvant (CFA) and other adjuvants used in research and veterinary applications have toxicities that limit their potential use in human vaccines. New chemically defined preparations such as QS-21, muramyl dipeptide, and monophosphoryl lipid A are being studied.

The messenger RNAs present in HeLa cells late in adenovirus 2 infection have been characterized utilizing three complementary methods of analysis. In each case the mRNAs were defined functionally, by identification of the polypeptides they encode when translated in a reticulocyte cell-free system. The RNAs were examined by (1) electrophoretic fractionation in agarose gels containing methyl-mercury hydroxide to estimate the sizes of the functional RNAs; (2) selection by annealing to viral DNA fragments immobilized on nitrocellulose to define the genomic origin of their constituent sequences; (3) hybrid-arrested translation using viral DNA fragments to locate their protein coding sequence. The results allow the positioning of both the coding and non-coding regions of all the late mRNAs with respect to the DNA. They demonstrate that the mature mRNAs originating in the major late transcription unit fall into five families. Within each family the mRNAs overlap in a staggered fashion, sharing the same 3′ terminus but differing at their 5′ termini. The data also reveal the existence of two new late proteins of 52,000 and 55,000 Mr encoded between map co-ordinates 30 to 34 and permit the positioning of the late RNAs, according to the polypeptides they encode, as follows: 52, 55K · IIIa: III · pVII · V: pVI · II: 100 K/33 K · pVIII: IV. (The colons signify the ends of families; centre dots the junctions within families; the slash an overlap of coding sequences; and the comma, coding sequences which have not been separated in this analysis. A 55 K protein has Mr = 55,000.) Those mRNAs which are either the only transcripts from a specific region (such as those for polypeptides IX, IVa2 and IV) or are the 3′-terminal transcripts within a family (such as those coding for polypeptides IIIa, V, II and pVIII) migrate as discrete species upon electrophoresis in agarose gels containing methyl-mercury hydroxide. However, the 5′-proximal RNAs within a family (such as those encoding polypeptides 52, 55 K, III, pVII, pVI, 100 K and 33 K) exhibit a broad distribution of molecular weights.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

The Epstein-Barr virus (EBV)-specified polypeptides induced upon viral replication in the P3HR-1 cell line have been examined by immunoprecipitation with a high-titer human anti-EBV serum. Twenty-five predominant polypeptides were identified in cell extracts, whereas 18 polypeptides were precipitated from cell-free translation reactions directed by total mRNA. Hybrid selection of mRNA to the BamHI DNA clones of the EBV genome and immunoprecipitation of the corresponding cell-free translation products revealed 98 EBV-specified polypeptides and their coding location along the viral genome. In addition, the viral polypeptides that bind reversibly to DNA-cellulose have been characterized and the deduced map locations of this functional group of EBV-specified polypeptides is presented.

Eight cases of lymphoid malignancy preceded by Bell's palsy are reported. There was a disproportionate number of ALL cases, two of which were of T-cell type. The possible pathogenic association of these conditions is discussed.

Simian adenovirus 7 (SA7) is a highly oncogenic virus, capable of causing tumors in hamsters upon the direct injection of viral DNA. We determined the transcriptional organization of the transforming region and compared it with that of the human adenoviruses. This analysis demonstrated that there are two independently promoted transcription units similar to the E1a and E1b regions of the human adenoviruses. The nucleotide sequence of the SA7 E1a region demonstrated considerable homology with the human adenoviruses, both in the sequences that regulate E1a expression and in the encoded polypeptides. The amino acid homology was reflected in the ability of SA7 to complement the growth of human adenoviruses mutant in the E1a region. Furthermore, we found two regions of amino acid homology unique to SA7 and the highly oncogenic human adenovirus 12.

We describe a simple procedure for isolating specific mRNAs and for mapping them to the regions of the DNA from which they originate. The method involves hybridization of total cytoplasmic RNA to restriction fragments of DNA that have been fractionated in agarose gels and immobilized on nitrocellulose filters. The hybridization-selected RNAs are eluted and translated in a cell-free system in order to identify their encoded polypeptides. Optimal hybridization and filter washing conditions are given for selection of mRNAs from adenovirus 2-infected cells and transformed cells.

Cytoplasmic polyadenylylated messenger RNA from differentiated muscle cultures, when incubated in a wheat germ cell-free system, directed the synthesis of a polypeptide indistinguishable from authentic chicken skeletal muscle actin, as judged by mobility on sodium dodecyl sulfate-polyacrylamide gels, tryptic peptide analyses, and biological activity. The synthesis of actin in the cell-free system was used to assay levels of translatable actin mRNA in cultures of fibroblasts, pre- and post-fusion myoblasts, and myoblasts grown under conditions that prevent fusion. In all cases the amount of actin polypeptide synthesized in the cell-free system was proportional to the rate of actin synthesis in the cultures from which the RNA was extracted. It is suggested that actin synthesis is regulated by the actin mRNA content of the cell and that an increase in the cytoplasmic level of translatable actin messenger RNA is mediated by cell fusion rather than by the terminal round of DNA synthesis.

When rye embryos imbibe water they rapidly return to a condition of biochemical and structural complexity. Three stages of imbibition can be recognised: Phase I a short period (10 min) of physical wetting; Phase II a longer period (1 h) when little further imbibition occurs, followed by Phase III a continuous phase of active water uptake. The latter coincides with an increase in respiration rate and an increase both in the number of mitochondria and of cristae within them. Changes in fine structure become evident in all organelles in Phase III, after 2 h of imbibition. In the unimbibed embryo endoplasmic reticulum is present only as short crescents associated with electron lucent bodies, but in Phase III the endoplasmic reticulum proliferates to form many surrounding cirlets. After 6 h these circlets become fewer and instead the endoplasmic reticulum is seen in close association with the nuclear membrane. Concurrently incorporation of radioactive uridine and thymidine is first detectible. This suggests that the large increase in protein synthesis occurs on new ribosomes present on the reticulum associated with the nuclear membrane. For the first 6 h protein synthesis must occur either on polysomes within the dense packing of ribosomes or on these circlets of endoplasmic reticulum associated with electron lucent bodies.