Synthesis of New Amido-Derivatives of 5-Fluorouracil and their Cytotoxic Activity against Various Continuous Cell Lines

Insect contractile cells frequently appear at an early phase of cell culture, but in most cases, they disappear before a continuous cell line is established, so the cell line ceases to contract. Continuous contractile insect cell lines are currently available from only one species each of Hymenoptera and Diptera. Here, we obtained a new cell line that contracted long after being established as a continuous cell line. The cell line contracted for a short period at an early phase of insect cell culture before a continuous cell line was established, but then did not contract again for several years. After this cell line entered the continuous growth phase, it produced spontaneously contractile tissues for about 4 mo but stopped contracting again. This is the first instance of a cell line that contracted after its establishment as a non-contractile continuous cell line. It is unclear whether the contractile cells survive or die after contraction ceases at an early phase of cell culture, and our results indicate that potential contractile cells survive for years after they stop to contract. The cells of this line sometimes produced complex contractilestructures, such as sheet-like tissues. Only a few continuous cell lines have been derived from scarabaeid beetles. The new continuous cell line was derived from the culture of the fat bodies of the scarab beetle Anomala cuprea, which is a pest in the agriculture and forestry of Japan. The population doubling time of the new cell line was 2.5 d and thus it grows very rapidly among coleopteran continuous cell lines. Our new cell line will facilitate research on the physiology and pathology of Coleoptera, including scarab beetles, and may also contribute to research on invertebrate muscles.

Pheochromocytomas are rare tumours, with an incidence of 1-2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line (KNA) was established from a sporadic pheochromocytoma of the right adrenal gland of a 73-year-old woman. The KNA cells grow as suspensions of spheroids and show the morphological and immunocytochemical characteristics of neuronal chromaffin cells, such as neuroendocrine granules, and positive reactions to chromogranin- and related peptide-, neuron specific enolase and vasoactive intestinal peptide antibodies. Neurite-like processes are formed after addition of nerve growth factor. Chromosomal analyses revealed a diploid (46,XX,n = 50) to hypodiploid (43-45,XX,n = 15) karyotype. In hypodiploid metaphases most frequently #19, #17, #21 and #22 were missing. Chromosome arms 1p and 4q showed apparently consistent interstitial deletions: 6q, 8q, 13q and 22q showed clonal interstitial deletions. The cell line shows a heterozygous sequence variant TGC (cysteine) to TGG (tryptophan) in codon 611 in exon 10 of the RET proto-oncogene. So far, PC-12, a rat adrenal pheochromocytoma, has been the only continuous pheochromocytoma cell line available. KNA represents the first report on a human continuous pheochromocytoma cell line, the first report of structural chromosome aberrations in pheochromocytomas and the first report of a RET mutation TGC to TGG in exon 10 of the RET proto-oncogene in a sporadic pheochromocytoma.

Discussion and Summary A continuous cell line derived from kidney tissue of the African green monkey, Cercopithecus aethiops and designated BS-C-1, has been maintained through 142 subcultures. Cultures of the BS-C-1 line support the growth of simian virus 40 to the same extent as do primary Cercopithecus kidney cell cultures; moreover, the pathognomonic vacuolating changes induced by this virus are as apparent in the continuous cell line as in the primary cell cultures. The BS-C-1 cell cultures are suitable for propagating several viruses, including polio, measles, Rift Valley fever, respiratory syncytial, Coxsackie A9, O'Malley's A-1 agent, and simian agents 1, 4 and 5. In contrast, several strains of influenza A and B viruses and of adenoviruses 3, 4 and 7 as well as simian virus 2 failed to multiply in BS-C-1 cells. No change in the susceptibility of the BS-C-1 cultures of SV40 virus was detected throughout 142 passages of the cell line. On the other hand, during this period of culture the cell line changed its chromosomal characteristics. Thus, the pattern remained diploid, i.e., 60 chromosomes, beyond the 20th subculture, and became subdiploid by the 41st passage after which it continued to change its characteristics with a majority of the cells in the 52nd passage having 59 chromosomes and the remainder 58; most of the cells had a dicentric chromosome. By the 111th passage, most of the cells approached the hypotetraploid state with 83% of the cells having 114 to 117 chromosomes. To our knowledge, this represents the first instance in which continuous observations have been made on the susceptibility of a tissue culture cell system to a given virus during a period when its cells changed from the diploid to the polyploid state. In this particular system the changes in chromosomal pattern were not associated with change in susceptibility to the virus. The BS-C-1 line is apparently free of extraneous microbial agents; seed cultures can be maintained in the frozen state; it grows rapidly and is not fastidious in its nutritional requirements. The BS-C-1 continuous cell line provides the virologist with another tool for diagnostic and research work and perhaps for the large scale cultivation of viral agents for vaccines. As regards the last mentioned possibility the employment of continuous cell lines has recently received a cautious endorsement (22) for use in carefully planned, progressively expanding studies.

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.

Hemopoietic stem cells may give rise to progeny like themselves or undergo determination; this event is followed by a series of maturation divisions ending in proliferatively inert but functional cells. In normal hemopoiesis and acute leukemia stem cell renewal is not exact; proliferative capacity is lost gradually. As a consequence, clonal populations cannot be continued indefinitely. Postdeterministic differentiation normally leads to cellular diversity; following transformation this diversity is increased, with the production of blast cells together with one or more myelopoietic lineage. The blasts are heterogeneous both in their proliferative capacity and their phenotypes, as determined using immunologically defined markers. Both self-renewal and determination are considered to be irreversible in vivo. By contrast, in continuous myelopoietic cell lines self-renewal is sufficiently precise to confer immortality on the populations. Furthermore, both determination and renewal may in some instances be reversible. The differences between normal or leukemic hemopoiesis in vivo and continuous lines in culture limits the value of the latter for studies of normal blood formation or the clonal hemopathies.

Immortalized and transduced cell lines are traditionally used in model of the HIV-1 life cycle. Primary cells may better represent the tissue of origin and events in vivo. We utilized an HIV-1/murine leukemia A4070 pseudotype virus and human Cyclin T1 to replicate HIV-1 in primary murine cells, and demonstrate that primary murine cells support HIV-1 infection better than immortalized cells.

Establishment of an erythroid cell line from primary CD36 + erythroid progenitor cells

The Cowden strain of porcine group C rotavirus (pararotavirus) was adapted to serial passage in a continuous monkey kidney cell line (MA104). Key factors in its successful adaptation included use of virus passaged in primary porcine kidney cells as the initial inoculum, use of roller tubes, and addition of pancreatin to the maintenance medium. A cell culture immunofluorescence test was used to quantitate the virus at each passage level, since a possible cytopathic effect was obscured by the effects of pancreatin. The virus titers dropped after initial passage into MA104 cells but increased thereafter, with peak titers evident after 16 passages (10(7) immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotavirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culture-propagated group C rotavirus was identical to that of the gut virulent virus from which it was derived. The cell culture-passaged group C rotavirus also retained its infectivity for gnotobiotic pigs. No group A rotavirus was detected in the intestinal contents of the pigs or in cell culture fluids from group C rotavirus-inoculated monolayers with the two former techniques or the cell culture immunofluorescence test. This is the first verified report of serial propagation of a non-group A rotavirus in a continuous cell line.

Ah receptor mediating induction of cytochrome P450IA1 in a novel continuous human liver cell line (Mz-Hep-1): Detection by binding with [ 3H]2,3,7,8-tetrachlorodibenzo- p-dioxin and relationship to the activity of aryl hydrocarbon hydroxylase

Previously published data suggest that the RGD-recognizing integrin, alphavbeta3, known as the vitronectin receptor, acts as a cellular receptor for RGD-containing enteroviruses, coxsackievirus A9 (CAV-9) and echovirus 9 (E-9), in several continuous cell lines as well as in primary human Langerhans' islets. As this receptor is also capable of binding the ligands by a non-RGD-dependent mechanism, we investigated whether vitronectin receptors, alpha v integrins, might act as receptors for other echoviruses that do not have the RGD motif. Blocking experiments with polyclonal anti-alphavbeta3 antibody showed that both primary human islets and a continuous laboratory cell line of green monkey kidney origin (GMK) are protected similarly from the adverse effects of several non-RGD-containing echovirus (E-7, -11, -25, -30, -32) infections. In contrast, corresponding studies on primary human endothelial cells showed that the receptor works only for E-25, E-30, E-32 and CAV-9. The inhibitory effect of the antibody was not restricted to prototype strains of echoviruses, as GMK cells infected with several field isolates of the corresponding serotypes were also protected from virus-induced cytopathic effects. Co-localization of virus particles with the receptor molecules in both GMK and primary human endothelial cells was demonstrated by live-cell stainings and confocal microscopy. Remarkably, in spite of similar virus-receptor co-localization and a comparable protective effect of the alphavbeta3 antibody, the entry pathways of the studied virus strains seemed to be divergent.

Continuous hematopoietic cell lines as model systems for leukemia–lymphoma research

This chapter describes the culture of various continuous cell lines. Continuous cell lines are generated either due to specific genetic or epigenetic alterations in the genome of certain cells. The reasons for genetic alterations are the manifestation of spontaneous or induced mutations via radiations and chemical or biological agents such as viruses. In the experimental laboratory conditions, continuous cell lines can be generated by a viral infection, biochemical agents, or radiations. This chapter discusses the culture of major mammalian continuous/cancer cell lines, including breast cancer cell line (MCF-7 and MDA-MB-231 cell), ovarian cancer cell line (CHO cells), prostate cancer cell line (LNCaP and PC3 cells), cancer cell lines originating from the female cervix (HeLa cells), lung cancer cell lines (A549 and BEAS-2B cells), kidney cancer cell lines (HEK-293 and VERO cells), liver cancer cell lines (HepG2 cells), and blood cancer cell lines (JURKAT and RAW cells). While some of the abovementioned cancer cell lines are isolated directly from various cancer patients, others are experimentally generated in the laboratory. The knowledge gained from this chapter would help to understand the culture and regular maintenance of the abovementioned continuous cell lines and in the development of various anticancer agents.KeywordsCulture of Continuous /Cancer Cell LinesMCF-7 Breast Cancer Cell LineMDA-MB-231 Breast Cancer Cell LineCHO Ovarian Cancer Cell LineLNCaP Prostate Cancer Cell LinePC3 Prostate Cancer Cell LineHeLa Cervix Cancer Cell LineA549 Lung Cancer Cell LineBEAS-2B Lung Cancer Cell LinesHEK-293 Kidney Cancer Cell LinesVERO Kidney Cancer Cell LineHepG2 Liver Cancer Cell LineJURKAT Blood Cancer Cell LinesRAW Blood Cancer Cell Lines

Preparation of immortalized cell lines obtained from organs and tissues of farm animals is an essential area of biotechnology. The paper presents results of continuous (immortalized) cell line preparation from a primary trypsinized cell culture of an adult rabbit kidney. Cytomorphologic analysis and karyotyping were performed during the process of subcultivation in the cell culture at passages 1, 3, 24, 31, 38, 56, 66, 75, 86, 101. Dynamics of spontaneous continuous cell line formation during long-term serial passaging was examined using standard nutrient media and fetal serum. Contrary to the known cell lines of rabbit origin (Oryctolagus cuniculus L.), immortalization was not accompanied with enhanced cell production and cell size reduction. The prepared continuous cell line in its adhesive phase was up to 200 µm in size and its productivity was about 7,000 cells/cm2. Significant differences (compared to the known cell lines) in the karyotype were detected during passaging. The formed genotype was found to be near-tetrapioid when the CCL cultural properties were stabilized at passages 66–101. The known cell lines – rabbit kidney (RK-13) and rabbit cornea (SIRC) – can be characterized as pseudotriploid basing on their karyotype. This culture demonstrated low sensitivity to viruses – causative agents of rabbit diseases and sensitivity to heterologous porcine and bovine viruses.

Rapidly growing primary cultures of normal human mammary epithelial cells (HMEC) were exposed to 1 microgram of benzo[a]pyrene (B[a]P) per ml for two or three 24-hr periods. The B[a]P-treated populations consistently contained cells displaying a longer period of active growth in culture compared to the untreated control cells. Widespread heterogeneity in morphology and growth patterns was evidenced in these "extended life" (EL) cultures, with multiple sequential changes in these parameters occurring during the course of their life in culture. Two apparently immortal continuous cell lines have thus far emerged from these EL cultures. These lines have been characterized to be of human mammary epithelial origin and derived from the originally treated HMEC specimen. The continuous lines do not appear to be malignantly transformed as they do not cause tumor formation in nude mice and show little or no anchorage-independent growth. Nonetheless, they have acquired several properties characteristic of tumor-derived HMEC, which distinguish them from their normal progenitors. These cell lines, as well as the EL strains, may provide useful substrates for studies to determine what agents can induce further transforming events. Additionally, analysis of the multiple steps occurring in the El cultures, as well as in the emergence of the continuous cell lines, could potentially elucidate the processes occurring during human epithelial cell carcinogenesis and escape from senescence.

Aim. To use the ability of potato leafroll virus (PLRV) to infect and multiply in mammalian continuous cell lines to purify PLRV isolates from the vegetative plant material, and to study the pathogenicity of those isolates for plants (after culturing in mammalian continuous cell line), to investigate morphological, physical-chemical, biological and antigen properties of PLRV isolates from mammalian cells and to study an alternative diagnostic method – the neutralization test in the mammalian continuous cell lines. Methods. The methods of cultivating animal viruses in the mammalian continuous cell line, microscopical biochemical, and serological methods, the method of artifi cial nutrition of aphids are detailed under Material and Methods. Results. It was demonstrated that successful cultivation of PLRV in mammalian continuous cell line allowed obtaining pure virus isolates from potato plants and aphids and preserving them for a long time (over a period of 7 years). The cultivation of PLRV in the mammalian continuous cell line did not impact its pathogenic properties and allowed transmitting the virus to plants. Continuous cells lines of pig embryonic kidney (PEKV), of kidney Syrian hamster (BHK- 21), of testicles of piglets (PTP), of kidneys of the bull (MDBC), and of carcinoma rabbit kidney (RK-13) were found to be sensitive to PLRV, Con tinuous cell lines of human (HeLa, Hep-2 and of African green monkey kidney (Vero) were not infected by the virus. The infectious activity of PLRV in the sensitive continuous cell lines was 20–8.5 lg TCD 50 /ml depending on the cell line. The isolates of PLRV were resistant to lipid- dissolving solvents, multiplied in a pH range from 4.0 till 10.0 and were thermoresistant at 50 oС in the absence of bivalent ions of magnesium, ТIP was in the range of 60–65 oС under our experimental conditions. The optimal temperature for the reproduction of PLRV in the cell culture was c. 24 °С. The use of neutralization test in the mammalian continuous cell line allowed isolation in pure culture and identifi cation of PLRV reliably in a time span of c. 14 days. Conclusions. It was proven that PLRV can be cultivated in the mammalian continuous cell lines of PEKV, ВНК-21, PTV, MDВК and RK-13. It was established that the cultivation of PLRV in these continuous cell lines did not impact its biological, pathogenic, antigenic and physical-chemical properties. The identifi cation of pure cultures of PLRV obtained in mammalian cells can be reliably performed by the use of neutralization reaction.