PD-1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction.

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8+ T cells are considered a barrier to immunosuppression-mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8+ T lymphocytes. Here we demonstrate that costimulation blockade-mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD-1) expression on CD8+ T cells. In the absence of PD-1 expression, CD8+ T cells form prolonged interactions with graft-infiltrating CD11c+ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.

Radiofrequency electromagnetic fields do not alter the cell cycle progression of C3H 10T and U87MG cells.

The effects of exposure to radiofrequency electromagnetic fields (RF EMFs) on cell cycle progression of mouse fibroblasts C3H 10T(1/2) and human glioma U87MG cells were determined by the flow cytometric bromodeoxyuridine pulse-chase method. Cells were exposed to a frequency-modulated continuous wave at 835.62 MHz or a code division multiple access RF EMF centered on 847.74 MHz at an average specific absorption rate of 0.6 W/kg. Five cell cycle parameters, including the transit of cells through G(1), G(2) and S phase and the probability of cell division, were examined immediately after the cells were placed in the fields or after they had been kept in the fields for up to 100 h. The only significant change observed in the study was that associated with C3H 10T(1/2) cell cultures moving into plateau phase toward the later times in the long-exposure experiment. No changes in the cell cycle parameters were observed in cells exposed to either mode of RF EMFs when compared to sham-exposed cells in either of the cell lines studied during the entire experimental period. The results show that exposure to RF EMFs, at the frequencies and power tested, does not have any effect on cell progression in vitro.

Multiple alternative splicing forms of human RAD17 and their differential response to ionizing radiation.

In this study, we have identified four alternatively spliced RAD17 RNAs, FM1, FM2, FM3, and FM4, which are produced through alternative splicing within the first 300 base-pairs of the coding region. FM3 and FM4 are two novel forms that have not been reported before. All four alternatively spliced RAD17 RNAs were detected in the tissues we examined. However, the levels of these forms varied from tissue to tissue. The expression of these four forms was also found to differ in different phases of the cell cycle and following exposure to X-irradiation. FM2, FM1, FM4, and FM3 encode putative polypeptides consisting of 681, 670, 596, and 516 amino acids, respectively. To determine if these polypeptides were expressed in cells, we generated a polyclonal antibody using a synthetic peptide. A major band around 71 kDa and two minor bands around 73 and 62 kDa were detected in human normal fibroblasts on Western blots. These three bands appear to represent the proteins encoded by FM2 (the 73 kDa band), FM1 (the 71 kDa band), and FM4 (the 62 kDa band) since the apparent molecular weights are close to their theoretical weights of the predicted amino acid sequences. The abundance of the 71 kDa protein was not significantly affected by X-irradiation, while the abundance of the 73 and the 62 kDa proteins was increased at least 5-fold 14 h postirradiation. The differential expression of these four alternatively spliced forms in different tissues, in different phases of the cell cycle, and their differential response to X-irradiation suggest that they may perform different functions in cell-cycle regulation and in the response to irradiation.

An alternative method to stereotactic inoculation of transplantable brain tumours in large numbers of rats.

The rat 9L gliosarcoma brain tumour model has been widely used in brain cancer studies. Intracerebral implantation of the cells in the parietal lobe of the brain has been performed using the stereotactic or freehand inoculation methods. For large numbers of rats, we wished to develop a method more accurate and precise than the freehand method, but less labour intensive than the stereotactic method. A template implantation technique was developed and compared quantitatively with the stereotactic method. Rats were inoculated with either the template or stereotactic method at doses of 1000, 5000, 10000, 20000 or 40000 cells. Results of this comparison showed that the template method is precise and accurate for tumour placement within the brain cortex, and decreases labour requirements. Mean survival rates between groups were not significantly different at doses of 5000, 20000 or 40000 cells inoculated. Significance was seen at the low dose of 1000 cells (P < 0.001). This was attributable to an absence of tumour growth in five of six stereotactic rats in this group. Significance was also seen at the 10000 dose level (P < 0.05) with the stereotactic rats again surviving longer than the template rats. However, in this case all the stereotactic rats had tumour growth. Brain weights did not differ significantly between groups, except at the 1000 dose level where no growth of tumour occurred in five of the six stereotactic animals. Body weight gain within one week following surgery did not differ significantly between any of the groups at alpha = 0.05. Studies on rat cadavers showed no statistical difference in placement measurements between the stereotactic and template methods. These results indicate that the template method for intracerebrally implanting tumour cells in rats provides a precise, accurate and rapid procedure that maximizes reproducibility with a significant reduction in labour requirements, when compared with the conventional stereotactic methodology.

Cell cycle-coupled variation in topoisomerase IIalpha mRNA is regulated by the 3'-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation.

Mammalian topoisomerase IIalpha (Topo II) is a highly regulated enzyme essential for many cellular processes including the G(2) cell cycle checkpoint. Because Topo II gene expression is regulated posttranscriptionally during the cell cycle, we investigated the possible role of the 3'-untranslated region (3'-UTR) in controlling Topo II mRNA accumulation. Reporter assays in stably transfected cells demonstrated that, similar to endogenous Topo II mRNA levels, the mRNA levels of reporter genes containing the Topo II 3'-UTR varied during the cell cycle and were maximal in S and G(2)/M relative to G(1). Topo II 3'-UTR sequence analysis and RNA-protein binding assays identified a 177-nucleotide (base pairs 4772-4949) region containing an AUUUUUA motif sufficient for protein binding. Multiple proteins (84, 70, 44, and 37 kDa) bound this region, and the binding of 84- and 37-kDa (tentatively identified as the adenosine- or uridine-rich element-binding factor AUF1) proteins was enhanced in G(1), correlating with decreased Topo II mRNA levels. The binding activity was enhanced in cellular extracts or cells treated with thiol-reducing agents, and increased binding correlated with decreased Topo II mRNA levels. These results support the hypothesis that cell cycle-coupled Topo II gene expression is regulated by interaction of the 3'-UTR with redox-sensitive protein complexes.

Radiofrequency electromagnetic fields have no effect on the in vivo proliferation of the 9L brain tumor.

The intracranial 9L tumor model was used to determine if exposure to a radiofrequency (RF) electromagnetic field similar to those used in cellular telephone has any effects on the growth of a central nervous system tumor. Fischer 344 rats implanted with different numbers of 9L gliosarcoma cells were exposed to 835.62 MHz frequency-modulated continuous wave (FMCW) or 847.74 MHz code division multiple access (CDMA) RF field with nominal slot-average specific absorption rates in the brain of 0.75 +/- 0.25 W/kg. The animals were exposed to the RF field for 4 h a day, 5 days a week starting 4 weeks prior to and up to 150 days after the implantation of tumor cells. Among sham-exposed animals injected with 2 to 10 viable cells (group 1), the median survival was 70 days, with 27% of the animals surviving at 150 days. The median survival length and final survival fraction for animals injected with 11 to 36 viable cells (group 2) were 52 days and 14%, respectively, while the values for those injected with 37 to 100 cells (group 3) were 45 days and 0%. The animals exposed to CDMA or FMCW had similar survival parameters, and the statistical comparison of the survival curves for each of the groups 1, 2 and 3 showed no significant differences compared to sham-exposed controls.

Thermal radiosensitization of human tumour cell lines with different sensitivities to 41.1 degrees C.

While much work on radiosensitization by hyperthermia in the 43 degrees C and higher temperature range has been done, relatively little work has been done at temperatures in the 41-42 degrees C range. In this moderate hyperthermia range there are dramatic differences in the resistance of mammalian cells to hyperthermia. Therefore, thermal radiosensitization was measured in two human colon adenocarcinoma cell lines, one that expresses chronic thermotolerance and proliferates at 41.1 degrees C, NSY 42129 (NSY) cells and one that is slowly killed at 41.1 degrees C, HCT15 cells. Heat-resistant NSY cells were found to be more radioresistant than heat-sensitive HCT15 cells. Hyperthermia at 41.1 degrees C enhanced the radiation sensitivity in NSY cells, but no significant induction of heat-induced radiosensitization was observed in HCT15 cells. The radiation sensitivity induced by 41.1 degrees C in NSY cells appeared to be related to both intrinsic heat-induced radiosensitization (HIR) and cell-cycle redistribution at 41.1 degrees C. Incidentally, cells incubated at 41.1 degrees C for between 8-16 h displayed an identical radiosensitivity to those heated for 24 h. This result implies that modest hyperthermia for 2 h or more can have a radiosensitizing effect in heat-resistant cells.

Intracellular distribution of hsp70 during long duration moderate hyperthermia.

Hyperthermia causes cell killing and is also an effective radiosensitizer. In recent years, the protocol of long duration moderate hyperthermia (LDMH) has been used to treat cancer patients in the clinic. However, the results of many studies indicate that some tumour cells may reveal the capability to express chronic thermotolerance, a factor of potentially critical impact in the efficacy of clinical hyperthermia. Previously it has been reported that two out of five human cell lines studied were able to proliferate at 41.1 degrees C. In the present study, the intracellular distribution of hsp70 during LDMH was measured as a potential marker for chronic thermotolerance with continued cell proliferation. In all cell lines studied, hsp70 became localized in the nucleus immediately after the cells were shifted from 37 degrees C to 41.1 degrees C. However, in the two cell lines which recovered and continued to proliferate, NSY42129 and HT29, hsp70 was delocalized from the nucleus within 4 h. Conversely, in the cell lines for which 41.1 degrees C was lethal, hsp70 did not delocalize from the nucleus but rather became localized in the nucleolar regions. Neither the NSY42129 cells nor the HT29 cells showed any preferential nucleolar punctate staining. Thus, it appears that the pattern of hsp70 nuclear localization and delocalization is related to the cells' ability to survive moderate heat shocks.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside.

OBJECT: Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promoters of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. METHODS: The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase-mediated biotinylated-deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. CONCLUSIONS: These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

A comparison of the modes and kinetics of heat-induced cell killing in HeLa and L5178Y cells.

The mode and kinetics of cell killing in HeLa and L5178Y cells were investigated to elucidate possible relationships between the mechanisms and modes of heat-induced cell death. L5178Y cells were heat-shocked for either 24 min at 43 degrees C or 30 min at 45 degrees C, while HeLa cells were given only the latter treatment. The degree of heat-induced excess nuclear protein correlated with cell death in HeLa cells but not in L5178Y cells. This difference suggests that the mechanism of cell death differs between these cell lines. With L5178Y cells the main mode of death after treatment at 43 degrees C was apoptosis with little progression of cells through the cell cycle. However, after treatment at 45 degrees C these cells died by necrosis without progressing through the cell cycle. HeLa cells were found to die by a mechanism other than apoptosis after significant progression of cells through the cell cycle and perturbation of the normal distribution of cells in the phases of the cell cycle (specifically, the fraction of cells in S and G2 phase increased 50% and 30%, respectively, prior to the occurrence of measurable cell death). These results are consistent with the hypothesis that the response to injury which has the potential to be lethal varies between different cell types, and results in the induction of different pathways leading to cell death.

Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside.

Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promotors of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 +/- 0.15% compared with 0.42 +/- 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 microM of cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 +/- 2.6% compared with 5.3 +/- 0.7% terminal deoxynucleotidyl transferase--mediated biotinylated--deoxyuridine triphosphate nick-end labeling technique-positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 microM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Carcinoma of the rectum. Possible cellular predictors of metastatic potential and response to radiation therapy.

BACKGROUND: Preoperative radiation therapy can markedly improve local control of rectal carcinoma. However, some tumors do not respond well to moderate doses of preoperative radiation and would be better served by more aggressive preoperative treatment (e.g., chemoradiotherapy). Cellular predictors of responsiveness to radiation can help to select lesions for more aggressive treatment. In addition, there is a need for cellular predictors of metastatic potential. This is particularly important in the setting of preoperative radiation-downstaging by preoperative treatment can obscure the true pathologic stage of a tumor and confound the usual selection criteria for postoperative chemotherapy. PURPOSE: This study was undertaken to determine if proliferating cell nuclear antigen (PCNA), p53, DNA ploidy, and S-phase fraction are associated with response to radiation and/or risk for distant metastatic disease and to determine if these cellular markers are best evaluated from preradiation biopsy specimen or the larger (but possibly altered) final surgical specimen. MATERIALS AND METHODS: Archival specimens from 23 cases of ultrasound T3 or T4 rectal carcinoma treated preoperatively with radiation therapy were reviewed. Eligible lesions had preradiation biopsy specimens of sufficient size for flow cytometric review of archival tissue. Factors considered included PCNA positivity, presence of mutant nuclear p53, more than 30 percent tumor cells in S-phase, and presence of aneuploidy. RESULTS: With a median follow-up of three years, overall freedom from relapse was 83 percent, with all but one failure being extrapelvic. PCNA positivity in the preradiation specimen was significantly (P = 0.025) associated with a greater risk of tumor recurrence. In addition, there was a trend to greater likelihood of "probable downstaging" (defined as surgical T stage less than preradiation ultrasound T stage) for lesions that were PCNA-negative or lesions with normal p53. Biomarkers measured in the postradiation surgical specimen were not associated with either freedom from relapse or response to radiation. Radiation treatment appeared to produce false-negatives in the final specimen. Thus, there were significantly more specimens converting from PCNA-positive to PCNA-negative after preoperative radiation than would be expected solely on the basis of sampling errors (P = 0.004). Similar results were found for abnormal p53 findings (P = 0.02). CONCLUSIONS: Prospective studies of biomarkers should be based on pretreatment specimens if preoperative radiation is given. For carcinoma of the rectum, PCNA and p53 may be useful predictors of both metastatic potential and responsiveness to radiation.

Chronic thermotolerance with continued cell proliferation.

The human colon adenocarcinoma cell line, NSY42129, is capable of proliferation at 41.1 degrees C. This ability appears to be due to a type of chronic thermotolerance, as opposed to selection or adaptation, that allows these cells to traverse S phase at elevated temperatures. Four other human cell lines were studied for their ability to proliferate at 41.1 degrees C. Of those only one, also a colon adenocarcinoma, showed the ability to sustain proliferation at 41.1 degrees C. While all the cell lines examined showed increased levels of the major heat shock proteins at 41.1 degrees C, the cellular amounts of these proteins did not correlate with their ability to proliferate at 41.1 degrees C. However, the ability of the cells to proliferate at 41.1 degrees C did correlate with their ability to sustain elevated rates of synthesis of hsp70 and hsp90. These results could have implications in the clinical application of hyperthermia, particularly the use of long duration moderate hyperthermia.

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression.

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd) -pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G2, i.e., the fraction of BrdUrd-negative G2 cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G2 and in S are equally delayed. S phase transit of cells irradiated in S or in G1 did not appear to be affected. There were apparent changes in flow of cells out of G1, which could be explained by the delayed entry of G2 cells into the compartment because of G2 arrest. Thus, in asynchronous cells the method was able to detect G2 delay in those cells irradiated in S and G2 phases and demonstrate the absence of cell-cycle delays in other phases.

Accelerated G1-transit following transient inhibition of DNA replication is dependent on two processes.

Accelerated G1 transit in HeLa cells develops after a temporary inhibition of DNA synthesis in the previous generation. G1 acceleration has been found to be dependent on (1) the cell-cycle position at the time of the inhibition and (2) the duration of the inhibition. The degree of G1 acceleration correlates with the position in S-phase at which DNA synthesis is blocked. G1 transit time decreases gradually beginning with early S blocks and reaches a minimum when DNA synthesis is inhibited at mid S-phase. A minimum of 4 h continuous inhibition at this position resulted in maximum G1 acceleration. Unbalanced growth, i.e., the nonspecific increase in total protein or RNA levels that results from inhibition of DNA synthesis, did not correlate with the decrease in G1 transit time. On the other hand, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed specific changes in four nuclear proteins of molecular weights 120, 66, 57, and 51 kDa in cells immediately following release from inhibition of DNA synthesis at mid S-phase. A Western blot analysis of cyclins D and E showed an increased accumulation of cyclin E at 0, 1, 5, 7, and 12 h following cell release from the mid S-phase block. Therefore, it is unlikely that changes in the ratio of protein/DNA or RNA/DNA are responsible for the decrease in G1 transit times as suggested by the unbalanced growth model of G1 acceleration. Instead, G1 acceleration may result from the accumulation during S-phase blockage, and subsequent retention through G2 and mitosis, of specific proteins required for cell transit through G1 in the next cell cycle.

Flow cytometric BrdUrd-pulse-chase study of heat-induced cell-cycle progression delays.

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell-cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43 degrees C for 5, 10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G1, G1S, and G2) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G1 compartment of approximately 16 min per minute of heat at 43 degrees C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43 degrees C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was approximately 25 min per minute of heat at 43 degrees C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G2 + M. The decrease in the rate of transit out of G2 for cells heated in G2 was significantly larger than that for any other compartment, consistent with previous studies, which showed a G2 accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.

Alterations in nuclear protein mass and macromolecular synthesis following heat shock.

Possible correlations between post-heat alterations in nuclear protein mass and the resumption of macromolecular (DNA, RNA, and protein) synthesis were investigated in CHO cells. Nuclear protein content was measured using flow cytometry. Macromolecular synthesis was measured by incorporation of radioactively labeled precursors into TCA-precipitable material of whole cells and isolated nuclei. Following an initial increase which was dependent on the heat dose, nuclear protein mass decreased following a monotonic function which appeared to be multiphasic. The synthesis of DNA, RNA, and protein was inhibited in a manner dependent on the heat dose and remained suppressed for an interval dependent on the heat dose before recovery. The kinetics of resumption of DNA, RNA, and protein synthesis correlated linearly with the nuclear protein mass measured immediately after heating. Also, the time of onset of recovery of RNA and protein syntheses correlated linearly with the time at which nuclear protein mass returned to 125% of control, a level which has been implicated as a possible threshold in previous studies. More significantly, the onset of the resumption of DNA synthesis showed a one-to-one correlation with the time at which the nuclear protein mass returned to 125% of control. These correlations suggest that there may be causal relationships between the resumption of DNA, RNA, and protein synthesis and the reduction of the amount of nuclear protein binding, particularly in the case of DNA synthesis.

The suppression of the synthesis of a nuclear protein in cells blocked in G2 phase: identification of NP-170 as topoisomerase II.

Previous studies of a nuclear protein of molecular weight 170 kDa (NP-170) have shown it to have two interesting properties. First, NP-170 synthesis began in mid- to late S phase and became maximal in G2 phase. Second, the synthesis of NP-170 was suppressed in cells blocked in G2 phase following irradiation with 6.8 Gy (J. M. Holland et al., Radiat. Res. 122, 197-208, 1990). The molecular weight of NP-170 is the same as that of Topoisomerase II (Topo II), an enzyme involved in the alteration of DNA supercoiling status with a double-strand passing function. This study was undertaken to determine whether NP-170 could be Topo II. The results from the present study show that both the proteins have identical cell cycle synthesis patterns. The synthesis of both these proteins is suppressed following irradiation. NP-170 was found to be recognized by a Topo II antibody in both Western blots and immunoprecipitation. This study characterizes NP-170 as Topo II.

G1 shortening following unbalanced growth: a specific v. nonspecific effect.

The synthesis and abundance of proteins were examined in synchronous populations of HeLa cells under conditions in which the lengthening of S phase, by inhibiting DNA synthesis, resulted in shortening of G1 in the subsequent generation. Mitotically collected cells were resynchronized by incubation with 3 microM aphidicolin from 3 to 12 h after mitotic selection; they were blocked again at various times thereafter to induce unbalanced growth. Cells were labelled with [35S]-methionine before and after release from the block to study the changes in protein synthesis. Triton X-100 soluble and insoluble proteins were analysed by 7-15% gradient SDS-PAGE, and radioactivity incorporation was quantified by liquid-scintillation counting. The degree of G1 shortening correlated with S phase position, increasing gradually from early S and reaching maximum when cells were blocked half-way through S phase. Synthesis of proteins of 120, 66, and 51 kDa was stimulated, and synthesis of a new protein of 57kDa was observed, in cells in which DNA synthesis had been blocked in mid-S. These proteins also showed increased accumulation. These results suggest that the shortening of G1, induced by the prior arrest of cell-cycle progression, is associated with synthesis of specific proteins rather than the non-specific accumulation of cellular proteins through unbalanced growth.