Changes in growth characteristics of mouse ascites tumor AISM during the process of passage in vivo

The growth characteristic of mouse ascites tumour AISM was observed on two distant passages (35th and 117th) in vivo. The following growth differences were established; the duration of life time of tumour bearing mice is less at the 35th passage (8 days) in comparison to the 117th passage (12 days); the common tumour cell mass at the terminal stage of life of tumour is more than 10 times less at 35th passage (10(8) cells) than at 117th passage (1.2.10(9) cells). The growth rate at 35th passage increases to the 4th day and at 117th passage to the 6th day. It is suggested that the tumour growth rate and the final size of tumor cell mass depend on the cell ploidity and chalone growth control.     

Long-term in vitro cell culture of Sinclair swine melanoma.

The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in cluture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell cultures of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18.     

Characterization of metabolic profiles and lipopolysaccharide effects on porcine vascular wall mesenchymal stem cells

The link between metabolic remodeling and stem cell fate is still unclear. To explore this topic, the metabolic profile of porcine vascular wall mesenchymal stem cells (pVW-MSCs) was investigated. At the first and second cell passages, pVW-MSCs exploit both glycolysis and cellular respiration to synthesize adenosine triphosphate (ATP), but in the subsequent (third to eighth) passages they do not show any mitochondrial ATP turnover. Interestingly, when the first passage pVW-MSCs are exposed to 0.1 or 10 μg/ml lipopolysaccharides (LPSs) for 4 hr, even if ATP synthesis is prevented, the spare respiratory capacity is retained and the glycolytic capacity is unaffected. In contrast, the exposure of pVW-MSCs at the fifth passage to 10 μg/ml LPS stimulates mitochondrial ATP synthesis. Flow cytometry rules out any reactive oxygen species (ROS) involvement in the LPS effects, thus suggesting that the pVW-MSC metabolic pattern is modulated by culture conditions via ROS-independent mechanisms.     

Long-term in vitro cell culture of sinclair swine melanoma

Summary The melanoma of Sinclair swine exhibits several characteristics similar to human melanoma but demonstrates an unusually high incidence of spontaneous regression. A total of 66 finite cell lines derived from 21 swine melanotic lesions, both cutaneous and visceral, were studied in vitro over their life spans of up to 14 months. The growth characteristics of the cultures varied with the age of the swine from which the tumors were obtained. Cell cultures of tumors obtained from swine aged less than 2 months grew steadily in culture with a population-doubling time of 120 to 180 hr until growth and division ceased after a maximum of 25 to 35 population doublings (6 to 8 passages). Cell culture of tumors obtained from swine aged 3 months or older showed a biphasic growth pattern with an early slow growth rate (population-doubling time 120 to 160 hr), which shifted after 3 to 6 passages to a faster rate (80 to 110 hr population-doubling time) until termination of growth and division after a maximum of 75 to 85 population doublings (18 to 20 passages). The cultures were morphologically heterogeneous including cuboidal, spindle and dendritic cell types. Electron microscopy showed classic melanosomes only in the primary and passage 1 cultures although vesicular inclusions were numerous in later-passage cells. However, continued melanin synthesis was indicated by the spectroscopic characteristics of material obtained from medium of passage 8 cultures and by DOPA staining of cultures as advanced as passage 18. This work was supported by a grant from the NIH, NCI (2 P01 CA 08023-11A1).     

Evolution of Sindbis Virus with a Low-Methionine-Resistant Phenotype Is Dependent Both on a Pre-Existing Mutation and on the Methionine Concentration in the Medium

SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium. Whether the passage was begun with SV319 or with SV321, the dominant virus population which emerged always acquired the second SVlm21 amino acid change. However, when the passage was begun with virus having neither the nt 319 or the nt321 mutation, even after many passages neither of these mutations was seen in the passaged virus population. Virus with the LMR phenotype emerged earlier when the virus encoded a wild-type RDRP (passage 4) rather than the mutant RDRP encoded by SVpzf (passage 7). When the methionine concentration in the medium of mosquito cells was increased to 250 µM, more than 20 passages were required until the LMR phenotype predominated. Competition experiments were carried out to compare the relative fitness of SVlm21, SVwt, SV319 and SV321 to each other. Our results indicated that SVlm21 was dominant to SVwt, as well as to both SV319 and SV321. However, SV319 and SV321 were able to co-exist with SVwt implying that in these mixed infection the presence of SVwt inhibited the emergence of SVlm21. Finally, our experiments highlight how a virus population by mutation and selection can adapt to the intracellular concentration of a simple metabolite, S-adenosylmethionine.     

Increase in Gs and cyclic AMP generation in HIT cells. Evidence that the 45-kDa alpha-subunit of Gs has greater functional activity than the 52-kDa alpha-subunit

Cyclic AMP accumulation in response to forskolin, cholera toxin, or isoproterenol is dramatically increased in HIT T-15 cells, a clonal cell line of Syrian hamster pancreatic islet beta cells, as a function of passage number. Forskolin and cholera toxin elevate cyclic AMP levels 5- to 10-fold higher in later passages (87-100) than in earlier passages (70-80). A similar phenomenon is observed with isoproterenol (10 microM) which increases cyclic AMP levels 56-fold in older HIT cells (passage 94), whereas only marginally stimulating cyclic AMP production in younger cells (passage 70-82). To determine whether a change in the stimulatory or inhibitory guanine nucleotide regulatory proteins, Gs or Gi, was responsible for these observations, ADP-ribosylation of HIT cell membranes with cholera toxin and pertussis toxin was examined. All passages contained two cholera toxin substrates at 52 and 45 kDa. The amount of 52 kDa did not appear to change with passage number, but the amount of 45 kDa increased in the later passages (89 and 94). The ratio of 45 to 52 kDa cholera toxin substrate, as determined by densitometric analysis, increased from 0.1 in passages 70, 75, and 82 to 0.45 at passage 89. No passage related changes in a 40-kDa pertussis toxin substrate were observed. An increase in the amount of the 45-kDa alpha-subunit of Gs was confirmed on immunoblots using antisera specific for the alpha-subunits of Gs. The amount of functional Gs present in various HIT cell passages was examined by determining the extent to which extracts from HIT cell membranes reconstituted guanine nucleotide-sensitive adenylyl cyclase in S49 cyc- membranes. Extracts derived from passage 94 reconstituted three to four times more adenylyl cyclase activity in cyc- membranes than extracts from passages 70, 75, and 82. These data indicate that an increase in functional Gs in later passages may be the underlying cause for the increased responsiveness to isoproterenol and forskolin in later passages. These data also suggest that functional differences exist between the Gs alpha-subunits, with the smaller 45-kDa subunit being more efficacious in coupling to cyclic AMP synthesis than the larger 52-kDa subunit. This is a departure from the commonly held view that the two subunits have similar efficacies in stimulating adenylyl cyclase.     

Establishment of a cell line (SH-4) from pleural effusion of a patient with melanoma.

A continuous tissue culture line (SH-4) derived from pleural effusion cells of a patient with metastatic melanoma is described. The cells became established after five passages lasting a period of 5 months of slow growth. Doubling time of the continuous culture was 20 hr in passage 51. The cell line is now in passage 60. The cells of all passages examined appear spindle-shaped or oligodendritic as seen by light microscopy. Pigment deposition in the cells is light and incomplete, but more marked than in cells of the original pleural effusion as shown by electron microscopy. Chromosome complement shows a single peak with a modal number of 51.     

Human fibroblasts from normal and malignant breast tissue grown in vitro show a distinct senescence profile and telomerase activity

The telomerase activity and the senescence profile of cultured breast fibroblasts from normal human interstitial and malignant stromal tissue were studied in comparison with their proliferation and differentiation pattern. Fibroblasts were grown either in the presence or absence of a conditioned medium (CM) obtained from cultures of the oestrogen receptor-positive breast cancer MCF-7 cell line. At different passages (from the 2nd up to the 48th), fibroblasts were examined for the telomerase activity by the Telomerase Repeats Amplification Protocol (TRAP) assay, for proliferation profile by Ki-67 antigen expression, and the myofibroblast or smooth muscle cell-like differentiation pattern by immunofluorescence with monoclonal antibodies specific for smooth muscle markers. Serial passages of fibroblasts from normal or tumour breast reveal that the relationship between the levels of telomerase activity and phenotypic/proliferation profile changes with cell subcultivation in a different manner in the two cell populations. The fibroblasts from normal tissue completed 12 passages in a CM-independent way prior to senescence whereas fibroblasts from tumour stroma senescence were attained after 48 passages. These cells showed a marked decrease of telomerase activity, growth rate and smooth muscle -actin expressing myofibroblasts after the 32nd passage. CM treatment of this fibroblast population induces a decline in the myofibroblast content, which precedes the changes in telomerase activity. Passaged fibroblasts from normal breast tissue can be converted to myofibroblasts upon CM treatment whereas those from tumour stroma were CM-insensitive. Taken together our data suggest that a heterogeneous fibroblast population with different life span is activated/recruited in the breast interstitium and poses the problem of a unique activation/recruitment of fibroblasts in neoplastic conditions.     

Genetic characterization of Sabin types 1 and 3 poliovaccine virus following serial passage in the human intestinal tract.

Poliovirus isolates types 1 and 3 were obtained from five and seven successive passages respectively, in infants who had been fed monovalent OPV in two separate clinical trials conducted in 1960. The purpose of these trials was to answer the question how much the vaccine virus would revert to its original neurovirulent phenotype following multiplication in the intestinal tract. Human passages were performed either by contact exposure or by feeding the excreted virus while the infants were maintained in isolation. Several virus isolates were obtained at each passage level. Infants participating in both studies showed no symptoms of disease. Antigenic studies (McBride, van Wezel) and protein analysis (PAGE) of the isolates, reported earlier from this laboratory, had shown that the isolates remained vaccine-like, although isolates from the later passages revealed some differences. Monkey neurovirulence test results showed that for both types 1 and 3 viruses the loss of attenuation of the vaccine strain upon passage was gradual, although the loss was faster for type 3. Examination of the oligonucleotide maps demonstrated that the oligonucleotide configuration of the isolates remained the same as for the vaccine strain but there was an increase of individual spot differences with increasing passage. The nucleotide sequence analysis of selected regions of the virus genomes revealed that there was no change from a G to A in nucleotide 480 of type 1 isolates; however, nucleotide 476 changed from a U to an A in type 1 passages 3, 4 and 5. Conversely, for type 3 the change of nucleotide 472 from a U to a C changed at the early first passage (4 days following administration of OPV), and remained a C in the six following passages; type 3 nucleotide 2034 did not change in the first passage from a U to a C, but it became a C in all further passages tested. The nucleotide changes mentioned for both virus types remained stable in successive passages. However, there was another nucleotide change for type 3 from a U to a C at position 1973 only for passages 5 and 6 which reverted to a U for passages 7L and 7LL. Study of selected human passage virus strains could further contribute to the identification of the critical nucleotides that are responsible for the attenuation of these two polio types of vaccine viruses.     

Change in the hybridization characteristics of rapidly-labelled ribonucleic acids during tumor "progression"]

The hybridization properties of in vivo rapidly labeled with 14C-orotate both nuclear and mitochondrial ribonucleic acids from the MD hepatoma were investigated. During tumour progression the repression of nuclear genome found at its early stages (5th to 6th passages) is replaced by the increase of hybridizability of nuclear DNA with a population of 14C-RNA's as well as by the appearance of new classes of pulse labeled RNA's. In other words, at late stages of tumour progression (60th passage) there occur a de-repression of nuclear genome. The hybridizability of mitochondrial RNA with nuclear DNA remains almost the same at different tumour progression stages. The results obtained are discussed in the light of literature data available.     

Phenotypes and Karyotypes of Human Malignant Mesothelioma Cell Lines

Background

Malignant mesothelioma is an aggressive tumour of serosal surfaces most commonly pleura. Characterised cell lines represent a valuable tool to study the biology of mesothelioma. The aim of this study was to develop and biologically characterise six malignant mesothelioma cell lines to evaluate their potential as models of human malignant mesothelioma.

Methods

Five lines were initiated from pleural biopsies, and one from pleural effusion of patients with histologically proven malignant mesothelioma. Mesothelial origin was assessed by standard morphology, Transmission Electron Microscopy (TEM) and immunocytochemistry. Growth characteristics were assayed using population doubling times. Spectral karyotyping was performed to assess chromosomal abnormalities. Authentication of donor specific derivation was undertaken by DNA fingerprinting using a panel of SNPs.

Results

Most of cell lines exhibited spindle cell shape, with some retaining stellate shapes. At passage 2 to 6 all lines stained positively for calretinin and cytokeratin 19, and demonstrated capacity for anchorage-independent growth. At passage 4 to 16, doubling times ranged from 30–72 hours, and on spectral karyotyping all lines exhibited numerical chromosomal abnormalities ranging from 41 to 113. Monosomy of chromosomes 8, 14, 22 or 17 was observed in three lines. One line displayed four different karyotypes at passage 8, but only one karyotype at passage 42, and another displayed polyploidy at passage 40 which was not present at early passages. At passages 5–17, TEM showed characteristic features of mesothelioma ultrastructure in all lines including microvilli and tight intercellular junctions.

Conclusion

These six cell lines exhibit varying cell morphology, a range of doubling times, and show diverse passage-dependent structural chromosomal changes observed in malignant tumours. However they retain characteristic immunocytochemical protein expression profiles of mesothelioma during maintenance in artificial culture systems. These characteristics support their potential as in vitro model systems for studying cellular, molecular and genetic aspects of mesothelioma.     

Trypanosoma cruzi: intracellular stages grown in a cell-free medium at 37 C.

A liquid medium containing a high concentration of water-soluble vitamins and ATP was developed for serial cultivation of Trypanosoma cruzi at 27–37 C; fetal bovine serum and trypticase were the only undefined substances in this medium. At 27 C, Trypanosoma cruzi grows primarily (over 99%) as epimastigotes with a population density reaching 92.7 × 10⁶/ml after 12 days of incubation. During the first subculture at 37 C, many epimastigotes from the original inocula changed into metacyclic trypomastigotes after 48 hr; the trypomastigotes subsequently transformed into amastigotes by 96 hr. In the second passage at 48 hr, 57.8% of the organisms were trypomastigotes which changed into amastigotes by the end of the incubation period. The proportion of amastigotes in the third and subsequent passages increased steadily as the proportion of epimastigotes gradually diminished. Amastigotes thus obtained could be serially subcultured indefinitely, yielding population densities of over 3.0 × 10⁷/ml of medium in 4–5 days at 37 C. Available evidence indicates that these amastigotes are morphologically and physiologically similar to intracellular amastigotes.     

申克孢子丝菌菌丝相至酵母相转化的实验研究

目的观察申克孢子丝菌菌丝相向酵母相转化的形态学变化并初步研究连续传代后菌株转化为酵母相的百分率。方法将95株申克孢子丝菌临床株于脑心浸液琼脂培养基上连续传代至酵母相,利用显微镜及血细胞计数板计数菌丝和孢子比例并记录显微镜下形态。结果 95株申克孢子丝菌中,经1次传代即成功转化有23株,经2次传代有14株,经3次传代有10株,经4次传代有6株,4次传代后总计55.8%实验菌株转化为酵母相。结论部分申克孢子丝菌由菌丝相向酵母相转化需经过连续多次传代,连续传代增加了菌丝相至酵母相的转化率。     

Growth behavior of human pancreatic carcinoma xenograft correlates with nuclear features.

Two human pancreatic ductal adenocarcinomas with different growth rates were serially transplanted into nude mice. Feulgen-stained 4 microns sections and imprints from the xenografts were studied with a VICOM automated image analysis system. After pooling the results from two passages, with three mice in each passage, it was shown that of 23 nuclear parameters measured the following were correlated with a fast tumor growth rate: in sections, a decrease in heterogeneity of the chromatin and an increase in perimeter and nuclear area; in imprints, an increase in lesser diameter, in mean grey level difference between second neighboring pixels, and in total integrated optical density (DNA content). Several parameters differed significantly between passages, and between animals in the same passage. These findings suggest that the growth speed of pancreatic tumors may be predicted by nuclear parameters.     

Growth properties and cholesterol removal ability of electroporated Lactobacillus acidophilus BT 1088

This study aimed to evaluate the effects of electroporation on the cell growth, cholesterol removal, and adherence abilities of L. acidophilus BT 1088 and their subsequent passages. The growth of electroporated parent cells increased (P<0.05) by 4.49-21.25% compared with that of the control. This may be attributed to the alteration of cellular membrane. However, growth of first, second, and third passages of treated cells was comparable with that of the control, which may be attributed to the resealing of transient pores on the cellular membrane. Electroporation also increased (P<0.05) assimilation of cholesterol by treated parent cells (>185.40%) and first passage (>21.72%) compared with that of the control. Meanwhile, incorporation of cholesterol into the cellular membrane was also increased (P<0.05) in the treated parent cells (>108.33%) and first passage (>26.67%), accompanied by increased ratio of cholesterol:phospholipids (C:P) in these passages. Such increased ratio was also supported by increased enrichment of cholesterol in the hydrophilic heads, hydrophobic tails, and the interface regions of the membrane phospholipids of both parent and first passage cells compared with that of the control. However, such traits were not inherited by the subsequent second and third passages. Parent cells also showed decreased intestinal adherence ability (P<0.05; decreased by 1.45%) compared with that of the control, without inheritance by subsequent passages of treated cells. Our data suggest that electoporation could be a potential physical treatment to enhance the cholesterol removal ability of lactobacilli that was inherited by the first passage of treated cells without affecting their intestinal adherence ability.     

Expression of epidermal differentiation antigens in cultures of interspecific somatic hybrids (human keratinocytes X mouse fibroblasts 3T3-4E)

Interspecific somatic hybrids have been prepared by fusion of human epidermal cells with mouse fibroblasts 3T3-4E using PEG 4000. Expression of epidermal differentiation antigens (bullous pemphigoid antigens, BP, keratin subsets 55-57 k and 67 k), markers of basal and suprabasal cells, were studied by immunocytochemistry for 10 passages. These markers were detected in the hybrids early after fusion, indicating that cells from both compartments were able to fuse with 3T3-4E cells. However, the hybrids expressing high molecular weight keratins were no longer detected after 7 days in primary cultures and serial passages, whereas those expressing BP antigens and vimentin persisted. Low molecular weight keratins 52 K and 50 K were detected by SDS-PAGE at the second passage in precipitates formed between labeled hybrid lysates and total keratin rabbit antiserum. Karyotype analysis showed mainly murine chromosomes and a submetacentric human chromosome between the 6th and the 10th passage.     

Reciprocity Versus Rhythmicity in Spontaneous Alternations of Nasal Airflow

Active anterior rhinomanometry was used to observe nasal airflow in five men and four women (ages 18-30). Measurements were obtained for each nasal passage every 5 min throughout an uninterrupted 8-hr session. Facial skin temperature from the left and right side of the face was recorded simultaneously from thermocouples. Observations were made during the months of May and June; subjects were allowed to maintain their routine diurnally active schedules prior to observation. Airflow in the two passages showed a significant negative correlation (i.e. Was reciprocal) in 44% of subjects (N= 9). Autocorrelation and spectral analysis of the airflow data found evidence of periodicity in 39% of individual nostrils and 56% of subjects. Mean estimated period was 4.5 ± 1.0 hr (range 3.5-6.0 hr). Only 22% of subjects showed statistical evidence of periodicity in both nostrils (i.e. a “nasal cycle”). Left- and right-side facial skin temperatures changed in parallel rather than reciprocally, but showed evidence of periodicity in 50% of hemifacial time series (56% of subjects), with an estimated period of 3.8 ± 1.0 hr (range 2.3-5.0).     

Reciprocity Versus Rhythmicity in Spontaneous Alternations of Nasal Airflow

Active anterior rhinomanometry was used to observe nasal airflow in five men and four women (ages 18–30). Measurements were obtained for each nasal passage every 5 min throughout an uninterrupted 8-hr session. Facial skin temperature from the left and right side of the face was recorded simultaneously from thermocouples. Observations were made during the months of May and June; subjects were allowed to maintain their routine diurnally active schedules prior to observation. Airflow in the two passages showed a significant negative correlation (i.e. Was reciprocal) in 44% of subjects (N= 9). Autocorrelation and spectral analysis of the airflow data found evidence of periodicity in 39% of individual nostrils and 56% of subjects. Mean estimated period was 4.5 ± 1.0 hr (range 3.5–6.0 hr). Only 22% of subjects showed statistical evidence of periodicity in both nostrils (i.e. a “nasal cycle”). Left- and right-side facial skin temperatures changed in parallel rather than reciprocally, but showed evidence of periodicity in 50% of hemifacial time series (56% of subjects), with an estimated period of 3.8 ± 1.0 hr (range 2.3–5.0).