scholarly journals Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product.

1986 ◽  
Vol 6 (5) ◽  
pp. 1386-1392 ◽  
Author(s):  
J P Durkin ◽  
J F Whitfield
Keyword(s):  
Protein Synthesis ◽  
Rat Kidney ◽  
Temperature Sensitive ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ras Gene ◽  
Serum Free ◽  
P21 Protein ◽  
Conditioning Factors ◽  
Sarcoma Virus

NRK rat kidney cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus (ts371) were transformed at 36 degrees C but were phenotypically nontransformed at 41 degrees C because of the abnormal thermolability of the oncogenic 21-kilodalton product of the viral Ki-ras gene. Thus tsK-NRK cells were rendered quiescent in a G0-G1 state by a 48-h incubation in serum-free medium at the nonpermissive, p21-inactivating temperature of 41 degrees C. The serum-starved cells could then be stimulated to transit G1 either as nontransformed cells by adding serum at 41 degrees C or as transformed cells by lowering the temperature to a p21-activating 36 degrees C. The viral p21 protein was as effective as serum in stimulating tsK-NRK cells to transit G1 and to start replicating DNA. While p21 effectively stimulated cells to transit G1 even in unconditioned, serum-free medium, they still needed cell-derived conditioning factors to subsequently divide. The p21 protein also enabled the cells to transit G1 in spite of an extracellular Ca2+ deficiency that inhibited the G1 transit of serum-stimulated cells. p21 activity was needed to stimulate both early and late G1 events. In contrast to serum, p21 did not stimulate total RNA or protein synthesis, but some RNA and protein synthesis must have been needed for the p21-driven G1 transit because it could be stopped by actinomycin D or cycloheximide.

Characterization of G1 transit induced by the mitogenic-oncogenic viral Ki-ras gene product

1986 ◽  
Vol 6 (5) ◽  
pp. 1386-1392
Author(s):  
J P Durkin ◽  
J F Whitfield
Keyword(s):  
Protein Synthesis ◽  
Rat Kidney ◽  
Temperature Sensitive ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ras Gene ◽  
Serum Free ◽  
P21 Protein ◽  
Conditioning Factors ◽  
Sarcoma Virus

NRK rat kidney cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus (ts371) were transformed at 36 degrees C but were phenotypically nontransformed at 41 degrees C because of the abnormal thermolability of the oncogenic 21-kilodalton product of the viral Ki-ras gene. Thus tsK-NRK cells were rendered quiescent in a G0-G1 state by a 48-h incubation in serum-free medium at the nonpermissive, p21-inactivating temperature of 41 degrees C. The serum-starved cells could then be stimulated to transit G1 either as nontransformed cells by adding serum at 41 degrees C or as transformed cells by lowering the temperature to a p21-activating 36 degrees C. The viral p21 protein was as effective as serum in stimulating tsK-NRK cells to transit G1 and to start replicating DNA. While p21 effectively stimulated cells to transit G1 even in unconditioned, serum-free medium, they still needed cell-derived conditioning factors to subsequently divide. The p21 protein also enabled the cells to transit G1 in spite of an extracellular Ca2+ deficiency that inhibited the G1 transit of serum-stimulated cells. p21 activity was needed to stimulate both early and late G1 events. In contrast to serum, p21 did not stimulate total RNA or protein synthesis, but some RNA and protein synthesis must have been needed for the p21-driven G1 transit because it could be stopped by actinomycin D or cycloheximide.

The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium

1987 ◽  
Vol 7 (1) ◽  
pp. 444-449
Author(s):  
J P Durkin ◽  
J F Whitfield
Keyword(s):  
S Phase ◽  
Temperature Sensitive ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ras Gene ◽  
Serum Free ◽  
Quiescent Cells ◽  
Sarcoma Virus ◽  
G2 Phase ◽  
Oncogene Products

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.

scholarly journals The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.

1987 ◽  
Vol 7 (1) ◽  
pp. 444-449 ◽  
Author(s):  
J P Durkin ◽  
J F Whitfield
Keyword(s):  
S Phase ◽  
Temperature Sensitive ◽  
Serum Free Medium ◽  
Free Medium ◽  
Ras Gene ◽  
Serum Free ◽  
Quiescent Cells ◽  
Sarcoma Virus ◽  
G2 Phase ◽  
Oncogene Products

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.

scholarly journals Induction of apoptosis by c-Fos protein.

1996 ◽  
Vol 16 (1) ◽  
pp. 211-218 ◽  
Author(s):  
G A Preston ◽  
T T Lyon ◽  
Y Yin ◽  
J E Lang ◽  
G Solomon ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Colorectal Carcinoma ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Fos Protein ◽  
Induced Apoptosis ◽  
I Cells

The role of c-Fos in apoptosis was examined in two Syrian hamster embryo cell lines (sup+I and sup-II) and a human colorectal carcinoma cell line (RKO), using the chimeric Fos-estrogen receptor fusion protein c-FosER. As previously reported, contrasting responses were observed when these two cell lines were placed under growth factor deprivation conditions; sup+I cells were highly susceptible to apoptosis, whereas sup-II cells were resistant. In this report, we show that the activated c-FosER protein induces apoptosis in sup-II preneoplastic cells in serum-free medium, indicating that c-Fos protein can induce apoptotic cell death in these cells. c-Fos-induced apoptosis was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that the c-Fos transcriptional activation activity is not involved. This conclusion was further supported by the observation that overexpression of v-Fos, which is highly proficient in transcriptional activation but deficient in the transcriptional repression activity associated with c-Fos, did not induce apoptosis. Constitutively expressed Bcl-2 delayed the onset of low-serum-induced apoptosis in sup+I cells and enhanced survival in sup-II cells. Further, coexpression of Bcl-2 and c-FosER in sup+I or sup-II cells protected the cells from c-FosER-induced apoptosis. The possibility that c-FosER-induced apoptosis requires a p53 function was examined. Colorectal carcinoma RKOp53+/+ cells, which do not normally undergo apoptosis in serum-free medium, showed apoptotic DNA fragmentation upon expression and activation of c-FosER. Further, when the wild-type p53 protein was diminished in the RKO cells by infection with the papillomavirus E6 gene, subsequent c-FosER-induced apoptosis was blocked. The data suggest that c-Fos protein plays a causal role in the activation of apoptosis in a p53-dependent manner. This activity does not require new protein synthesis and is blocked by overexpression of Bcl-2 protein.

scholarly journals Cell-cycle-specific induction of quiescence achieved by limited inhibition of protein synthesis: counteractive effect of addition of purified growth factors

1985 ◽  
Vol 73 (1) ◽  
pp. 375-387
Author(s):  
O. Larsson ◽  
A. Zetterberg ◽  
W. Engstrom
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Growth Factors ◽  
Growth Factor ◽  
Serum Starvation ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
G1 Cells ◽  
Synthesis And Degradation

We have previously shown that Swiss 3T3 cells located in the first part of G1 (post-mitotic G1 cells younger than 4.0 h or G1pm cells) were arrested after 9–10 h in the cell cycle by a short (1-8 h) exposure to serum-free medium or by a short (2-4 h) exposure to low doses of the protein synthesis inhibitor cycloheximide (CH). Kinetic data indicate that such G1pm cells rapidly return to G0 during this brief treatment and thereafter require a preparatory period of 8 h before continuing to G1. Cells older than 4 h, i.e. cells in mid or late G1 are already committed to DNA synthesis (presynthesis or G1ps cells). These cells as well as S and G2 cells were consequently unaffected by the brief serum starvation or the brief treatment with cycloheximide. In the present paper we show that the 10-h intermitotic delay that follows a 1–2 h exposure to serum-free medium can be completely counteracted by the presence of any one of the purified growth factors, epidermal growth factor (EGF), insulin or platelet-derived growth factor (PDGF). In contrast, the intermitotic delay following a longer exposure (8 h) to serum-free medium could no longer be counteracted by EGF or insulin. However, PDGF was still active in this respect. Most interestingly, the 12 h gross intermitotic delay induced by a 4h exposure to CH could be efficiently counteracted by EGF, PDGF or insulin. However, this effect on CH-treated cells could be counteracted by the growth factor only in the presence of 10% serum. This indicates the existence of a cooperative effect between PDGF, EGF or insulin and an unidentified serum factor. The effects on the cell cycle time of brief serum starvation and exposure to CH were compared with the effects on rate of protein synthesis and degradation. Although the effects of serum starvation on protein synthesis and degradation were found to be partially normalized by growth factors, we suggest that growth factors prevent cells from leaving the cell cycle by another mechanism and not merely by affecting the level of overall protein accumulation.

Exogeneous factors are not necessary in attachment, spreading and polarization of hela cells

1978 ◽  
Vol 36 (2) ◽  
pp. 310-311
Author(s):  
W. Liebrich
Keyword(s):  
Hela Cells ◽  
Calf Serum ◽  
Glass Tube ◽  
Serum Free Medium ◽  
Nacl Solution ◽  
Free Medium ◽  
Minimum Essential Medium ◽  
Serum Free ◽  
All Solutions ◽  
Glass Support

HeLa cells were grown for 2-3 days in EAGLE'S minimum essential medium with 10% calf serum (S-MEM; Seromed, München) and then incubated for 24 hours in serum free medium (MEM). After detaching the cells with a solution of 0. 14 % EDTA and 0. 07 % trypsin (Difco, 1 : 250) they were suspended in various solutions (S-MEM = control, MEM, buffered salt solutions with or without Me++ions, 0. 9 % NaCl solution) and allowed to settle on glass tube slips (Leighton-tubes). After 5, 10, 15, 20, 25, 30, 1 45, 60 minutes 2, 3, 4, 5 hours cells were prepared for scanning electron microscopy as described by Paweletz and Schroeter. The preparations were examined in a Jeol SEM (JSM-U3) at 25 KV without tilting.The suspended spherical HeLa cells are able to adhere to the glass support in all solutions. The rate of attachment, however, is faster in solutions without serum than in the control. The latter is in agreement with the findings of other authors.

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