Populations Of HeLa Cells Research Articles

The supply of exogenous deoxyribonucleotides accelerates the speed of the replication fork in early S-phase.

Earlier studies have established that the average speed of a replication fork is two to three times slower in early S-phase than in late S-phase and that the intracellular 2'-deoxyribonucleoside 5'-triphosphate pools grow during S-phase. In this study, the effect of the exogenous 2'-deoxyribonucleoside 5'-triphosphate (dNTP) supply on the average replication speed in a synchronised population of human HeLa cells was tested. The speed of replication fork movement was measured on extended DNA fibers labelled with 2'-deoxythymidine analogues 5-chloro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine. We show that the introduction of exogenous dNTPs accelerates the replication process at the beginning of DNA synthesis only. In late S-phase, the administration of additional dNTPs has no effect on the speed of replication forks. The availability of 2'-deoxynucleotides seems to be a rate-limiting factor for DNA replication during early S-phase.

Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18

Aims—To assess the validity and practicality of real time polymerase chain reaction (PCR) for human papillomavirus (HPV) testing in combination with liquid based cytology samples for cervical screening. Methods—Real time...

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Characterization of the Transport Properties of Cloned Rat Multidrug Resistance-associated Protein 3 (MRP3)

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta-D-glucuronide ([3H]E217betaG) and glucuronide of [14C] 6-hydroxy-5, 7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2, 4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217betaG were 67 +/- 14 microM and 415 +/- 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 +/- 0.7 microM and 3.4 +/- 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217betaG had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217betaG transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217betaG. The uptake of [3H]E217betaG was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.

Calcium/Calmodulin-dependent Phosphorylation and Activation of Human Cdc25-C at the G2/M Phase Transition in HeLa Cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).

Modulation in Cell Cycle and Cyclin B1 Expression in Irradiated HeLa Cells and Normal Human Skin Fibroblasts Treated with Staurosporine and Caffeine

The comparative effects of staurosporine or caffeine on G2-phase arrest and cyclin B1 expression in human skin fibroblasts (HSF) and transformed HeLa cells following γ-irradiation were examined by flow cytometry. Contrary to some earlier reports with HeLa cells, the arrest in G2after irradiation was accompanied by an increase in cyclin B1 levels in both asynchronous and synchronized HeLa cells irradiated in early S phase. Caffeine and staurosporine were equally effective in attenuating both the radiation-induced increase in cyclin B1 expression and the prolongation of G2in synchronous and asynchronous HeLa cell populations. Staurosporine treatment was less effective in down-regulating cyclin B1 expression in asynchronous HeLa cells at earlier time points following irradiation when compared to caffeine-treated cells. In synchronized HeLa cells, down-regulation of an irradiation-induced increase in cyclin B1 expression was similar to either staurosporine or caffeine treatments, with caffeine being more effective at later time points. An increase in cyclin B1 expression was also observed in irradiated HSF cells (synchronous and asynchronous), which decreased when the cells were treated with staurosporine or caffeine. However, staurosporine was ineffective in attenuating the radiation-induced prolongation of G2in synchronous and asynchronous HSF cells, whereas treatment of irradiated synchronous or asynchronous HSF cells with caffeine significantly reduced the prolongation of G2. These results suggest that both staurosporine and caffeine treatments act on different pathways of cell cycle control in normal and transformed cells, in terms of attenuation of G2block and diminution of elevated levels of cyclin B1 expression, in response to radiation.

Reconstitution of ATP-dependent Leukotriene C4 Transport by Co-expression of Both Half-molecules of Human Multidrug Resistance Protein in Insect Cells

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs is unknown. To facilitate studies on the structure and function of MRP, we have determined whether a baculovirus expression system can be used to produce active protein. Full-length MRP as well as molecules corresponding to either the NH2- or COOH-proximal halves of the protein were expressed individually and in combination in Spodoptera frugiperda Sf21 cells. High levels of intact and half-length proteins were detected in membrane vesicles from infected cells. Although underglycosylated, the full-length protein transported leukotriene C4 (LTC4) with kinetic parameters very similar to those of MRP produced in transfected HeLa cells. Neither half-molecule was able to transport LTC4. However, a functional transporter with characteristics similar to those of intact protein could be reconstituted when both half-molecules were co-expressed. Transport of LTC4 by Sf21 membrane vesicles containing either intact or reconstituted MRP was competitively inhibited by both S-decylglutathione and 17beta-estradiol 17-(beta-D-glucuronide), with Ki values similar to those reported previously for MRP expressed in HeLa cells (Loe, D. W., Almquist, K. C., Deeley, R. G., and Cole, S. P. C. (1996) J. Biol. Chem. 271, 9675-9682; Loe, D. W., Almquist, K. C., Cole, S. P. C., and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689). These studies demonstrate that human MRP produced in insect cells can function as an active transporter of LTC4 and that the NH2- and COOH-proximal halves of the protein can assemble efficiently to form a transporter with functional characteristics similar to those of the intact protein.

ATP-dependent 17β-Estradiol 17-(β-D-Glucuronide) Transport by Multidrug Resistance Protein (MRP): INHIBITION BY CHOLESTATIC STEROIDS

In addition to its ability to confer resistance to a range of natural product type chemotherapeutic agents, multidrug resistance protein (MRP) has been shown to transport the cysteinyl leukotriene, LTC4, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential physiological substrates also includes cholestatic glucuronidated steroids. ATP dependent, osmotically sensitive transport of the naturally occurring conjugated estrogen, 17 beta-estradiol 17-(beta-D-glucuronide) (E(2)17 beta G), was readily demonstrable in plasma membrane vesicles from populations of MRP-transfected HeLa cells (Vmax 1.4 nmol mg-1 min-1, K(m) 2.5 micron). The involvement of MRP was confirmed by demonstrating that transport was completely inhibited by a monoclonal antibody specific for an intracellular conformational epitope of the protein. MRP-mediated transport of LTC4, was competitively inhibited by E(2)17 beta G (K(i(app)) 22 micron), despite the lack of structural similarity between these two substrates. Competitive inhibition of [3H]E(2)17 beta G transport was also observed with a number of other cholestatic conjugated steroids. All of these compounds prevented photolabeling of MRP with [3H]LTC4, demonstrating that the cholestatic steroid and leukotriene conjugates compete either for the same or possibly overlapping sites on the protein. Consistent with the presence of overlapping but non-identical sites, studies using chemotherapeutic drugs to inhibit MRP-mediated E(2)17 beta G transport indicated that daunorubicin had the highest relative potency of the drugs tested, whereas it was the least potent inhibitor of LTC4 transport. Non-cholestatic steroids glucuronidated at the 3 position of the steroid nucleus, such as 17 beta-estradiol 3-(beta-D-glucuronide), did not compete for transport of E(2)17 beta G by MRP, nor did they inhibit photolabeling of the protein with [3H]LTC4. These data identify MRP as a potential transporter of cholestatic conjugated estrogens and demonstrate site-specific requirements for glucuronidation of the steroid nucleus.

All‐or‐nothing Ca2+ mobilization from the intracellular stores of single histamine‐stimulated HeLa cells.

1. Histamine-stimulated mobilization of intracellular Ca2+ stores was monitored in intact and permeabilized populations of HeLa cells using both the fluorescent Ca2+ indicator Fura-2 and 45Ca2+ measurements. Digital video imaging of Fura-2-loaded cells was used to measure the intracellular calcium concentration ([Ca2+]i) of single cells. 2. In populations of HeLa cells, histamine caused a concentration-dependent increase in cytoplasmic [Ca2+]. The initial transient increase was independent of extracellular Ca2+ (Ca2+o) and was followed by a sustained increase that was abolished by removal of Ca2+o. 3. In Ca(2+)-free medium ([Ca2+]o < 1 microM), a maximal histamine concentration (25 microM) caused a transient increase in [Ca2+]i, and a subsequent challenge with histamine failed to evoke a further response indicating that the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ stores had been completely emptied. Lower concentrations of histamine (0.5-10 microM) caused smaller, concentration-dependent increases in [Ca2+]i that were also transient. After exposure to these low histamine concentrations, where [Ca2+]i returned to baseline within 2 min, addition of a higher histamine concentration evoked a further increase in [Ca2+]i. The second increase in [Ca2+]i was inversely proportional to the increase caused by the first exposure to histamine, indicating that Ca2+ released in the initial response was not substantially resequestered into histamine-sensitive stores. 4. Single HeLa cells challenged with low concentrations of histamine in Ca(2+)-free medium responded with transient increases in [Ca2+]i, but individual cells differed in their sensitivity with 51% of cells responding to 1 microM, and 98% responding to 25 microM-histamine. 5. When single cells in Ca(2+)-free medium were challenged with stepwise increases in histamine concentration, they responded to each step with a transient [Ca2+]i increase after which [Ca2+]i returned to baseline within 1 min. Prolonging the interval between histamine additions by up to 25 min did not affect the [Ca2+]i increase evoked by a subsequent histamine addition. 6. Unidirectional 45Ca2+ efflux from saponin-permeabilized HeLa cells showed that, under conditions that prevented Ca2+ resequestration, submaximal concentrations of InsP3 rapidly emptied only a fraction of the InsP3-sensitive Ca2+ stores. The failure of low InsP3 concentrations to fully mobilize the InsP3-sensitive Ca2+ stores was not a consequence of InsP3 degradation. 7. We conclude that within single HeLa cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to InsP3, and the fraction of Ca2+ stores mobilized by InsP3 increases as the InsP3 concentration increases.

G1shortening 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.

Cell cycle regulation of an exogenous human poly(ADP‐ribose) polymerase cDNA introduced into murine cells

We have evaluated the regulation of expression of the poly(ADP-ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum-stimulated WI-38 cells, steady-state levels of the polymerase mRNA were highest at late S and S-G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP-ribose) polymerase cDNA under the control of a non-cell cycle-specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post-transcriptional level. The heterologous expression system confirmed that the post-transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP-ribose) polymerase may be autoregulated.

A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

A cis-Acting Element in the Promoter Region of the Murine c-myc Gene Is Necessary for Transcriptional Block

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

Lamin disassembly kinetics: A cell-free system with extracts from mitotic HeLa cells

We describe a cell-free system in which a postribosomal supernatant from metaphase HeLa cells induces prophase-like changes in permeabilized HeLa cell populations as evidenced by the nuclear lamin disassembly and chromatin condensation. We have attempted to characterize the cell-free system with permeabilized HeLa cells. First, by extracting lamins with agents known to disrupt the noncovalent interactions in the supramolecular lamin aggregate in interphase using polyclonal and a newly established monoclonal anti-lamin Ab 2E3, uniform extraction of lamins was achieved with urea and deoxycholate whereas the cation Mg 2+ and 2-mercaptoethanol had little effect on the disassembly of interphase lamins. Second, cytoplasmic extract from mitotic HeLa cells, synchronized by a nitrous oxide metaphase arrest, was tested. It had a differential effect on interphase lamin depolymerization. Nuclei in G 1 phase of the cell cycle were more resistant against the mitotic extracts than cells in S and G 2 phase. The results are discussed in terms of a possible inactivation of mitotic extracts by factors present in nuclei in early interphase.

Changes in heterogeneous nuclear RNP core polypeptide complements during the cell cycle.

Mammalian heterogeneous nuclear RNP (hnRNP) subcomplexes are shown to be comprised of 14-17 basic A and B core group polypeptides (chrp) when subjected to two-dimensional immunoblot analysis. These proteins are normally confined to the nucleus but are distributed throughout the cell during mitosis. However, not all of the 17 protein spots are observed for all stages of the cell cycle. HeLa cell populations have been synchronized and the basic hnRNP core protein complement examined during S, G2, mitosis, and G1. During cell division several distinct chrp polypeptide species at 35 and 37 kD appear, while another of 37 kD and a chrp of 38 kD are diminished. These altered chrp complements are not due to any effects induced by thymidine treatment but appear to be physiological changes in the chrp polypeptide modification state. The new charge isomers found during mitosis are not the result of selective phosphorylation of the chrp polypeptides. However the nature of the modifications has yet to be determined. The mitosis-specific modified forms of the chrp polypeptides are found in the cytoplasmic fraction derived from mitotic cell populations. When this fraction is centrifuged upon sucrose density gradients the modified chrp polypeptides sediment from 30-200S in a distribution similar to that of hnRNP complexes isolated from the nuclei of randomly dividing cell populations. RNase digestion experiments indicate that the general substructure of the RNA/protein complexes in mitotic cell cytoplasm is similar to that of nuclear hnRNP isolated from unsynchronized cells or tissue.

Expression of human HSP70 during the synthetic phase of the cell cycle.

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus E1A 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP70 mRNA rapidly increase 10- to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP70 expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP70 in the nucleus of replicating cells.

Transcription of the human hsp70 gene is induced by serum stimulation.

We have examined the expression of the heat shock protein (hsp70) gene in human cells. The transcription of the hsp70 gene and accumulation of cytoplasmic hsp70 mRNA is induced by serum stimulation. Populations of HeLa cells and human embryonic kidney cells (cell line 293) were serum starved. Upon serum stimulation, the level of hsp70 mRNA transiently increases between 12 and 18 hr to a 10-fold higher level. The increased levels of hsp70 mRNA can be accounted for by a 10- to 15-fold increase in the rate of transcription of the hsp70 gene. When cells were serum-stimulated in the presence of an inhibitor of DNA synthesis, 1-beta-D-arabinofuranosylcytosine (araC), the levels of hsp70 mRNA were induced to only 20% of the maximal level detected in the absence of the inhibitor. This suggests that the expression of the hsp70 gene is coupled to DNA synthesis. The cloned human hsp70 gene contains regulatory sequences that confer serum-stimulated transcriptional control. The endogenous hsp70 gene and the transfected chimeric gene containing sequences upstream of the hsp70 gene fused to bacterial chloramphenicol acetyltransferase are both temporally expressed in stable transfectants of cell line 293 cells. The endogenous hsp70 mRNA and the chimeric mRNA reach maximum levels 12-18 hr after serum stimulation.

Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cell cycle progression in HeLa cells.

Sodium butyrate alters the growth and gene expression of a variety of differentiating and neoplastic cell types. For example, addition of 5 mM butyrate to HeLa cells is reported to both induce gonadotropin alpha subunit biosynthesis and block cell cycling in G1. We have studied these two actions of butyrate on HeLa cells and found that they are regulated in distinct ways. The induction of alpha subunit synthesis was due to an increase in the rate of transcription of the alpha gene. Using synchronized populations of HeLa cells, we determined that butyrate stimulates alpha transcription throughout the cell cycle. In contrast, treated cells arrest in G1 only if exposed to butyrate for a discrete period during the previous S phase. We conclude that butyrate inhibits DNA synthesis through a cell cycle-specific action that is independent from its direct action to stimulate transcription of the gonadotropin alpha gene.

Independent regulation by sodium butyrate of gonadotropin alpha gene expression and cell cycle progression in HeLa cells.

Sodium butyrate alters the growth and gene expression of a variety of differentiating and neoplastic cell types. For example, addition of 5 mM butyrate to HeLa cells is reported to both induce gonadotropin alpha subunit biosynthesis and block cell cycling in G1. We have studied these two actions of butyrate on HeLa cells and found that they are regulated in distinct ways. The induction of alpha subunit synthesis was due to an increase in the rate of transcription of the alpha gene. Using synchronized populations of HeLa cells, we determined that butyrate stimulates alpha transcription throughout the cell cycle. In contrast, treated cells arrest in G1 only if exposed to butyrate for a discrete period during the previous S phase. We conclude that butyrate inhibits DNA synthesis through a cell cycle-specific action that is independent from its direct action to stimulate transcription of the gonadotropin alpha gene.

Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells

The intracellular localization of tumor antigens of human adenovirus type 5 (Ad5) during lytic infection of KB cells has been studied. The cells were pulse labeled with [ 35S]methionine early after infection and early proteins of 58,000 D (58K), 44K,19K,18.5K, and 14K detectable by immunoprecipitation with hamster antitumor serum were assayed for association with cytoplasm, nucleoplasm, chromatin, cytosol, cytoskeleton, and membranes. The 44,000 D (44K) tumor antigen encoded in early region IA (E1A: 0–4.4%) was recovered in approximately equal amounts from cytoplasmic and nucleoplasmic fractions of pulse-labeled cells and within the cytoplasmic compartment was found in the cytosol as well as associated with the cytoskeleton. The E1B-58K (E1B: 4.5–11.2%) antigen was also found to be associated with the cytoplasmic and nucleoplasmic fractions in approximately equal amounts but unlike the ElA-44K showed no affinity for cytoskeletons. Pulse-chase and immunofluorescence experiments suggested the 58K antigen accumulated in the nucleus late in infection. The E1B-19K antigen was found almost exclusively associated with the membrane fraction of infected KB cells and was resolved in polyacrylamide gels into two related species of 18.5K and 19K. Immunofluorescence studies on the ElB 18.5–19K doublet suggested that within a population of infected HeLa cells a small minority seemed to be expressing copious amounts of stainable antigen. Cell fractionation and immunofluorescence studies showed that the E4-14K antigen was a nuclear protein and the only antigen in this study which showed a significant association with a nuclear subfraction composed almost entirely of histones. The implications of these findings for the roles of the Ad5 tumor antigens in lytic infection and transformation are discussed.