scholarly journals Lactose synthetase activity in mouse mammary glands is controlled by thyroid hormones.

1979 ◽  
Vol 82 (3) ◽  
pp. 675-681 ◽  
Author(s):  
B K Vonderhaar
Keyword(s):  
Cell Cycle ◽  
Thyroid Hormones ◽  
Dna Synthesis ◽  
Milk Protein ◽  
Mammary Glands ◽  
Mouse Mammary Gland ◽  
The Other ◽  
Synthetase Activity

Epithelial cells in explants from the mammary glands of euthyroid mature virgin mice are proliferatively dormant. They must undergo DNA synthesis and traverse the cell cycle in vitro before they are able to differentiate fully in response to insulin, hydrocortisone, and prolactin, and synthesize enzymatically active alpha-lactalbumin (measured as lactose synthetase activity). In contrast, glands from hyperthyroid mature virgin mice do not require DNA synthesis in vitro to differentiate. Explants from the euthyroid virgin tissue overcome their dependence on DNA synthesis when 10(-9) M 3,5,3'-triiodo-L-thyronine is added directly to the cultures in addition to the other three hormones. Explants from involuted mammary glands from euthyroid primiparous mice do not require DNA synthesis in vitro to make the milk protein even though they, like explants from mature euthyroid virgin tissue, are proliferatively dormant and do not contain detectable lactose synthetase activity in vivo. Glands from primiparous animals made mildly hypothyroid by ingestion of 0.1% thiouracil in drinking water during 7 wk of involution remain morphologically indistinguishable from glands of their euthyroid counterparts. However, explants from the glands of these hypothyroid animals revert to a state of dependence on DNA synthesis to differentiate functionally. These observations suggest that the dependence on DNA synthesis and cell cycle traversal for hormonal induction of lactose synthetase activity in the mouse mammary gland is controlled by thyroid hormones.

Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

1996 ◽  
Vol 8 (6) ◽  
pp. 935 ◽  
Author(s):  
AW Schuetz ◽  
DG Whittingham ◽  
R Snowden
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Granulosa Cells ◽  
Dna Content ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Ovarian Follicles ◽  
S Phase

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

Cell cycle regulation of mouse H3 histone mRNA metabolism

1984 ◽  
Vol 4 (1) ◽  
pp. 123-132
Author(s):  
R B Alterman ◽  
S Ganguly ◽  
D H Schulze ◽  
W F Marzluff ◽  
C L Schildkraut ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Dna Synthesis ◽  
In Vitro Transcription ◽  
S Phase ◽  
Gene Probe ◽  
Histone Mrna ◽  
Mrna Content

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

CORRELATION BETWEEN THE EFFECTS OF HORMONES ON THE SYNTHESIS OF DNA IN EXPLANTS FROM INDUCED RAT MAMMARY TUMOURS AND THE GROWTH OF THE TUMOURS

1974 ◽  
Vol 62 (2) ◽  
pp. 225-240 ◽  
Author(s):  
D. LEWIS ◽  
R. C. HALLOWES
Keyword(s):  
Organ Culture ◽  
Dna Synthesis ◽  
Culture Medium ◽  
Mammary Glands ◽  
Sprague Dawley ◽  
Culture Techniques ◽  
Mammary Tumours ◽  
Sprague Dawley Rats

SUMMARY Explants from 32 mammary tumours induced in Sprague—Dawley rats by 9,10-dimethyl-1,2-benzanthracene (DMBA) were maintained in organ culture for up to 48 h. Insulin, corticosterone, prolactin, growth hormone and oestradiol were added to the culture medium in various combinations and their effects on the DNA synthesis of the explants was studied. DNA synthesis was stimulated by insulin in explants from 30 out of the 32 tumours examined and this group of 30 responsive tumours could be further subdivided. Explants from 16 tumours showed a greater rate of DNA synthesis in medium containing insulin plus corticosterone plus prolactin than in medium containing insulin alone and this higher rate was decreased by oestradiol; this group is referred to as 'prolactin-responsive'. Explants from the remaining 14 tumours did not show a greater rate of DNA synthesis in medium that contained insulin plus corticosterone plus prolactin than in medium containing insulin alone and neither rate was decreased by oestradiol; this group is referred to as 'insulin-responsive'. Explants from two tumours were not stimulated by insulin and these tumours are referred to as 'non-responsive'. After oophorectomy or administration of ergocryptine to tumour-bearing rats, the prolactin-responsive tumours regressed whereas the non-responsive tumours continued to grow. Explants taken from prolactin-responsive tumours 2 weeks after either oophorectomy or administration of ergocryptine were still prolactin-responsive but those taken from insulin-responsive tumours 2 weeks after the same treatment were now also prolactin-responsive. The non-responsive tumours remained non-responsive. The effects of hormones on the DNA synthesis in vitro of explants from growing DMBA-induced tumours were thus different from those on explants of mammary glands from virgin or pregnant Sprague—Dawley rats. It was concluded that it was possible to predict by organ culture techniques the response in vivo of growing mammary tumours to oophorectomy and ergocryptine administration.

scholarly journals Dissociation of cytological and functional differential in virgin mouse mammary gland during inhibition of DNA synthesis

1982 ◽  
Vol 53 (1) ◽  
pp. 97-114 ◽  
Author(s):  
B.K. Vonderhaar ◽  
G.H. Smith
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Dna Synthesis ◽  
Milk Protein ◽  
Light And Electron Microscopy ◽  
Mouse Mammary Gland ◽  
Enhanced Production ◽  
Virgin Mouse ◽  
In Cells

Epithelial cells in mammary gland explants from mice assume a secretory appearance and synthesize the milk proteins, casein and alpha-lactalbumin, when cultured in the presence of insulin, hydrocortisone and prolactin. In cells from the glands of mature virgin animals such syntheses are known to require DNA synthesis. Addition of cytosine-beta-D-arabinofuranoside to the explant cultures suppresses both hormonally induced DNA synthesis and enhanced production of milk protein. To determine the level at which this block in terminal differentiation occurs, epithelial cell pellets were prepared from virgin mouse mammary gland explants cultured with various combination of insulin, hydrocortisone and prolactin, and subsequently examined by light and electron microscopy. We observed that the epithelial cells cultured in the presence of all three hormones developed fully, cytologically and ultrastructurally, even in the absence of DNA synthesis in vitro. Likewise, these cells were able to incorporate [3H]uridine into RNA efficiently and to incorporate amino acids into acid-precipitable polypeptides at levels equivalent to the untreated controls. However, immunoprecipitation of newly synthesized casein peptides showed that no new synthesis of casein occurred in cells prevented from synthesizing DNA. These data show uncoupling of cytological development and synthesis of milk protein in mammary explants from mature virgin mice inhibited from synthesizing DNA.

scholarly journals Cell cycle regulation of mouse H3 histone mRNA metabolism.

1984 ◽  
Vol 4 (1) ◽  
pp. 123-132 ◽  
Author(s):  
R B Alterman ◽  
S Ganguly ◽  
D H Schulze ◽  
W F Marzluff ◽  
C L Schildkraut ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Steady State ◽  
Dna Synthesis ◽  
In Vitro Transcription ◽  
S Phase ◽  
Gene Probe ◽  
Histone Mrna ◽  
Mrna Content

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

scholarly journals Dihydroartemisinin Suppresses the Tumorigenesis and Cycle Progression of Colorectal Cancer by Targeting CDK1/CCNB1/PLK1 Signaling

2021 ◽  
Vol 11 ◽  
Author(s):  
You-Cai Yi ◽  
Rui Liang ◽  
Xiao-Yu Chen ◽  
Hui-Ning Fan ◽  
Ming Chen ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
Specific Inhibitor ◽  
Inhibitory Effects ◽  
Antitumor Effects ◽  
Cycle Progression ◽  
Cycle Arrest

Dihydroartemisinin (DHA), a well-known antimalarial drug, has been widely investigated for its antitumor effects in multiple malignancies. However, its effects and regulatory mechanisms in colorectal cancer (CRC) are still unproved. In this study, in vitro experiments including CCK8, EdU, Transwell, and flow cytometry analyses and an in vivo tumorigenesis model were conducted to assess the effects of DHA on the bio-behaviors of CRC cells. Additionally, RNA-seq combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses was used to obtain the targets of DHA, and these were verified by molecular docking, qRT-PCR, and Western blotting. As a result, we found that DHA significantly suppressed the proliferation, DNA synthesis, and invasive capabilities and induced cell apoptosis and cell cycle arrest in HCT116, DLD1, and RKO cells in vitro and in vivo. Further analyses indicated that the targets of DHA were predominantly enriched in cell cycle-associated pathways, including CDK1, CCNB1, and PLK1; and DHA could bind with the CDK1/CCNB1 complex and inhibit the activation of CDK1/CCNB1/PLK1 signaling. Moreover, cucurbitacin E, a specific inhibitor of the CDK1/CCNB1 axis, enhanced the inhibitory effects of DHA on DNA synthesis and colony formation in HCT116 and DLD1 cells. In short, DHA could suppress the tumorigenesis and cycle progression of CRC cells by targeting CDK1/CCNB1/PLK1 signaling.

scholarly journals Nafamostat mesilate, a nuclear factor kappa B inhibitor, enhances the antitumor action of radiotherapy on gallbladder cancer cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257019
Author(s):  
Naoki Takada ◽  
Hiroshi Sugano ◽  
Yoshihiro Shirai ◽  
Nobuhiro Saito ◽  
Ryoga Hamura ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Combination Treatment ◽  
Nuclear Factor Kappa B ◽  
The Other ◽  
Antitumor Action ◽  
Nafamostat Mesilate ◽  
Nuclear Factor ◽  
Radiation Induced

Nuclear factor kappa B (NF-κB) is a transcriptional factor that can be activated by radiotherapy and chemotherapy. The synthetic protease inhibitor nafamostat mesilate (NM) inhibits NF-κB activity and exerts antitumor actions in various types of cancer. In the present study, we hypothesized that NM might enhance the antitumor action of radiotherapy on gallbladder cancer (GBC) cells by inhibiting radiation-induced NF-κB activity. Thus, we investigated the correlation between radiotherapy and NF-κB activity in GBC cells. We assessed the in vitro effects of radiotherapy with or without NM on NF-κB activity, apoptosis of GBC cells (NOZ and OCUG-1), induction of apoptotic cascade, cell cycle progression, and viability of GBC cells using four treatment groups: 1) radiation (5 Gy) alone; 2) NM (80 μg/mL and 40 μg/mL, respectively) alone; 3) combination (radiation and NM); and 4) vehicle (control). The same experiments were performed in vivo using a xenograft GBC mouse model. In vitro, NM inhibited radiation-induced NF-κB activity. Combination treatment significantly attenuated cell viability and increased cell apoptosis and G2/M phase cell cycle arrest compared with those in the other groups for NOZ and OCUG-1 cells. Moreover, combination treatment upregulated the expression of apoptotic proteins compared with that after the other treatments. In vivo, NM improved the antitumor action of radiation and increased the population of Ki-67-positive cells. Overall, NM enhanced the antitumor action of radiotherapy on GBC cells by suppressing radiation-induced NF-κB activity. Thus, the combination of radiotherapy and NM may be useful for the treatment of locally advanced unresectable GBC.

scholarly journals Activation of the p34 CDC2 protein kinase at the start of S phase in the human cell cycle.

1992 ◽  
Vol 3 (4) ◽  
pp. 389-401 ◽  
Author(s):  
R L Marraccino ◽  
E J Firpo ◽  
J M Roberts
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin A ◽  
S Phase ◽  
Cdc2 Kinase ◽  
G1 Cells ◽  
Sv40 Dna ◽  
P34 Cdc2

Using a protocol for selecting cells on the basis of both size and age (with respect to the preceding mitosis), we isolated highly synchronous human G1 cells. With this procedure, we demonstrated that the p34 CDC2 kinase was activated at the start of S phase. Cyclin A synthesis began at the same time, and activation of the p34 CDC2 kinase at the start of S phase was, at least in part, due to its association with cyclin A. Furthermore, cells synchronized in late G1 by exposure to the drug mimosine contain active cyclin A/p34 CDC2 kinase, indicating that p34 CDC2 activation can occur before DNA synthesis begins. Thus, the cyclin A/CDC2 complex, which previously has been shown to be sufficient to start SV40 DNA synthesis in vitro, assembles and is activated at the start of S phase in vivo.

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo

1988 ◽  
Vol 8 (5) ◽  
pp. 1923-1931
Author(s):  
N H Heintz ◽  
B W Stillman
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
S Phase ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Restriction Fragments ◽  
Temporal Specificity

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.

scholarly journals Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

1988 ◽  
Vol 8 (5) ◽  
pp. 1923-1931 ◽  
Author(s):  
N H Heintz ◽  
B W Stillman
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
S Phase ◽  
Replication Forks ◽  
Chromosomal Dna ◽  
Restriction Fragments ◽  
Temporal Specificity

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.

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