Decreased Doubling Time Research Articles

Sustained in vitro trigger of self-renewal divisions in Hoxb4 hiPbx1 lo hematopoietic stem cells

Factors that trigger and sustain self-renewal divisions in tissue stem cells remain poorly characterized. By modulating the levels of Hoxb4 and its co-factor Pbxl in primary hematopoietic cells (Hoxb4hiPbxl(10) cells), we report an in vitro expansion of mouse hematopoietic stem cells (HSCs) by 105-fold over 2 weeks, with subsequent preservation of HSC properties. Clonal analyses of the hematopoietic system in recipients of expanded HSCs indicate that up to 70% of Hoxb4hiPbxl(10) stem cells present at initiation of culture underwent self-renewal in vitro. In this setting, Hoxb4 and its co-factor did not promote an increase in DNA synthesis, or a decrease in doubling time of Scal+Lin- cells when compared to controls. Q-PCR analyses further revealed a downregulation of Cdknlb (p27Kipl) and Mxdl (MadI) transcript levels in Hoxb4hiPbxl(l0) primitive cells, accompanied by a more subtle increase in c-myc and reduction in Ccnd3 (Cyclin D3). We thus put forward this strategy as an efficient in vitro HSC expansion tool, enabling a further step into the avenue of self-renewal molecular effectors.

Individual Overexpression of Five Subunits of Human Translation Initiation Factor eIF3 Promotes Malignant Transformation of Immortal Fibroblast Cells

Transcriptional and post-transcriptional regulatory mechanisms are commonly accepted paradigms of tumorigenesis. The view is emerging that deregulation of translation contributes importantly to cancer development, a role not generally appreciated before. Eukaryotic initiation factor eIF3 contains at least thirteen non-identical subunits, named from eIF3a to eIF3m, and plays an essential role in the rate-limiting initiation phase of translation. Increased mRNA and protein levels of the eIF3a, -3b, -3c, -3h, and -3i subunits have been detected in a wide variety of human tumors and are frequently identified as prognostic biomarkers for poor clinical outcome. However, it remains to be established whether up-regulation of eIF3 subunits is a consequence or a cause of the malignant phenotypes. Here we report that ectopic expression of eIF3a, -3b, -3c, -3h, or -3i in stably transfected NIH3T3 cells leads to a number of oncogenic properties: decreased doubling times, increased clonogenicity and viability, facilitated S-phase entry, attenuation of apoptosis, formation of transformed foci, and anchorage-independent growth. Only overexpression of the transforming subunits results in a stimulation of initiation and global protein synthesis rates and enhanced translation of poorly translated mRNAs that encode growth-regulating proteins, including cyclinD1, c-Myc, fibroblast growth factor-2, and ornithine decarboxylase, which may be responsible for oncogenic malignancy in the transformed cell lines. Based on these results, we hypothesize that eIF3 contributes to hyperactivation of the translation initiation machinery and thereby may play an important role in neoplasia. Cancer cells appear to require an aberrantly activated translational state to survive, suggesting that the initiation factors may be promising therapeutic targets for treating cancer.

EAR-2: Identification of a Gene Involved in Maintenance of Clonogenicity in Haematopoiesis.

Primary acute myelogenous leukaemia (AML) samples are heterogeneous in clonogenicity, both among patients and within the leukaemic cell population of a single patient. To explain this heterogeneity the leukaemia stem cell model postulates that leukaemic hematopoiesis is organized in a hierarchy, sustained by leukaemia stem cells that may either self-renew or differentiate aberrantly to give rise to blasts that can no longer proliferate. This process is akin to the irreversible growth arrest entered by terminally differentiating normal blood cells.We wished to identify genes associated with clonogenicity in AML. To obtain pure populations of cells of defined growth abilities, we analyzed low passage cultures of the cell line OCI-AML4. This cell line resembles primary AML cells in several important respects; it is growth factor-dependent, contains a low proportion of clonogenic cells, and has a relatively simple karyotype. Clones consisting of four cells were micromanipulated so that a single cell was sampled for global RT-PCR while its three clonal siblings served as reporters of clonogenicity. By microarray analysis we found the orphan nuclear receptor EAR-2 to be expressed four-fold lower in leukemia single cells that spontaneously lose proliferative ability, compared to single cells with greater proliferative capacity.EAR-2 is a member of the COUP transcription factor family, which play roles in various developmental processes through interactions with nuclear receptors and other transcription factors. We assessed expression of EAR-2 in monoblastic leukaemia U937 cells induced to differentiate with a variety of induction agents. Treatment with dimethylsulfoxide, phorbol ester, vitamin D3, and all trans retinoic acid (ATRA) all induced significant decreases in EAR-2 expression. This phenomenon was also seen in a mouse model of acute promyelocytic leukaemia (APL). When primary bone marrow cultures of hCG-NuMA-RAR transgenic mice were induced to differentiate with ATRA, an average decrement in EAR-2 expression of 5.58 fold was observed (p<0.005). Since aberrant differentiation is an invariant feature of AML, we hypothesized that the overall expression of EAR-2 would be greater in AML patients relative to healthy controls. Analysis by quantitative RT-PCR of 15 AML, 10 CMML, 12 MDS and 16 normal bone marrow samples showed that EAR-2 is overexpressed in all three disease categories (p<0.0009 AML, 0.03 CMML, 0.0003 MDS).To characterize the effect of forced expression of EAR-2 on clonogenicity we transduced U937 cells with a retrovirus encoding either EAR-2 (U937-EAR2) or EGFP (U937-GFP). Analysis of FACS-purified U937-EAR2 and U937-GFP cultures showed that forced expression of EAR-2 reduces the doubling time of these populations (U937-EAR2 = 24h; U937-GFP = 34h; p<< 0.001), while no significant difference was observed in cell cycle profile. The decrease in doubling time of U937-EAR2 cells may reflect a decrease in the rate of cell loss in the population, consistent with the hypothesis that EAR-2 functions as a repressor of terminal differentiation.We have observed that expression of the orphan nuclear receptor EAR-2 is positively associated with maintenance of proliferative capacity and negatively associated with differentiation. These observations establish the importance of EAR-2 in the regulation of clonogenicity and terminal differention.

Estrogen Receptor Expression and Sensitivity to Paclitaxel in Breast Cancer

A retrospective analysis of CALGB trial 9344 suggested paclitaxel administration following cyclophosphamide and doxorubicin adjuvant chemotherapy is most beneficial for patients with ER? negative (ER?-) breast cancer. Since the cytotoxic effects of paclitaxel are cell cycle dependent, we postulated that the relationship between ER? and the effectiveness of adjuvant paclitaxel reflects the observation that ER? positive (ER?+) breast cancers proliferate more slowly than ER?- breast cancers. Three in vitro models (MCF-7, T47D and ZR-75) were examined to compare growth rates and paclitaxel-induced apoptosis in ER?+ and ER?- clones of the same, originally ER?+ cell line. For the T47D and ZR-75 cell lines, loss of ER? was associated with a decrease in doubling time and an increase in paclitaxel sensitivity. However, when cell culture conditions were altered to achieve equivalent cell proliferation rates, no difference in paclitaxel sensitivity was observed. Similarly, an ER?- clone of MCF-7 cells that did not exhibit an enhanced growth rate compared to its ER?+ counterpart also did not show increased paclitaxel sensitivity. The combined apoptotic effects of tamoxifen and paclitaxel on MCF-7 cells were not synergistic or even clearly additive. In these in vitro models, the effectiveness of paclitaxel correlated more closely with growth rate than ER? expression. These data suggest that measurements of tumor proliferation may provide more accurate predictive markers for the benefits of adjuvant paclitaxel than ER? analysis.

Cyclin E deregulation alters the biologic properties of ovarian cancer cells.

We have previously shown that the low molecular weight (LMW) forms (trunk 1 and trunk 2) of cyclin E are biochemically hyperactive and induce G1/S progression in normal epithelial cells. Here we investigate the biologic consequences of LMW cyclin E expression in ovarian cancer cells. Using a panel of ovarian carcinoma tumors we find that cyclin E overexpression is invariably due to the presence of LMW forms and that expression of these forms appears to correlate with more advanced grade and stage of disease. Despite similar expression of p21 and p27, cyclin E overexpressing tumors have higher kinase function. Using an isogenic ovarian cancer model, we find that clones that overexpress the trunk 1 (T1) protein have a 10-fold increase in cyclin E kinase function, a 20% increase in S-phase fraction, a 10-15% decrease in doubling time and a 20% increase in colony formation compared to parental cells that express only the FL cyclin E protein. T1 clones were resistant to G1 arrest but more sensitive to cisplatin. Therefore, in ovarian tumors, the presence of LMW cyclin E forms confers altered biologic properties. Our data provides a potential mechanism for the poor prognosis of patients with LMW cyclin E expressing tumors.

Nerve growth factor and enhancement of proliferation, invasion, and tumorigenicity of pancreatic cancer cells

Nerve growth factor (NGF) exerts both stimulatory and inhibitory effects on neuronal and certain non-neuronal tumors. In pancreatic cancer NGF is overexpressed, and this overexpression is associated with increased perineural invasion. NGF has the potential to stimulate the growth of some pancreatic cancer cell lines, and this effect is mediated by the phosphorylation of tyrosine kinase receptor A and mitogen-activated protein kinase activation; it is dependent on the expression levels of tyrosine kinase receptor A and p75 receptors. To determine whether cancer cell-derived NGF can participate in the regulation of pancreatic cancer cell proliferation, PANC-1 human pancreatic cancer cells were stably transfected with a full-length human beta-NGF expression vector. In vitro and in vivo growth characteristics were analyzed by proliferation assays and invasion assays and in a nude mouse tumor model. Stable transfection of NGF in PANC-1 cells resulted in enhanced anchorage-dependent growth, with a decrease in doubling times of up to 50%, and in an approximately twofold increase in anchorage-independent cell growth and cell invasion. Furthermore, stably transfected PANC-1 cells showed enhanced tumorigenicity in nude mice. These results suggest that NGF has the capacity to act in a paracrine and/or an autocrine manner in pancreatic cancer and that it enhances cancer cell growth and invasion in vivo, thereby contributing to the aggressiveness and poor prognosis of this disease.

Germ line transmission of the Cdk4(R24C) mutation facilitates tumorigenesis and escape from cellular senescence.

Mutations in CDK4 and its key kinase inhibitor p16(INK4a) have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16(INK4a). To determine the role of the Cdk4(R24C) germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by using Cre-loxP-mediated "knock-in" technology. Cdk4(R24C/R24C) mouse embryo fibroblasts (MEFs) displayed increased Cdk4 kinase activity resulting in hyperphosphorylation of all three members of the Rb family, pRb, p107, and p130. MEFs derived from Cdk4(R24C/R24C) mice displayed decreased doubling times, escape from replicative senescence, and escape sensitivity to contact-induced growth arrest. These MEFs also exhibited a high degree of susceptibility to oncogene-induced transformation, suggesting that the Cdk4(R24C) mutation can serve as a primary event in the progression towards a fully transformed phenotype. In agreement with the in vitro data, homozygous Cdk4(R24C/R24C) mice developed tumors of various etiology within 8 to 10 months of their life span. The majority of these tumors were found in the pancreas, pituitary, brain, mammary tissue, and skin. In addition, Cdk4(R24C/R24C) mice showed extraordinary susceptibility to carcinogens and developed papillomas within the first 8 to 10 weeks following cutaneous application of the carcinogens 9,10-di-methyl-1,2-benz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). This report formally establishes that the activation of Cdk4 is sufficient to promote cancer in many tissues. The observation that a wide variety of tumors develop in mice harboring the Cdk4(R24C) mutation offers a genetic proof that Cdk4 activation may constitute a central event in the genesis of many types of cancers in addition to melanoma.

Opioid growth factor regulates the cell cycle of human neoplasias.

The native opioid growth factor (OGF), [Met5]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and migration, as well as tissue organization, during development, cancer, homeostatic cellular renewal, wound healing, and angiogenesis. OGF action is mediated by the OGF receptor (OGFr). To investigate the target of OGF as to cell proliferation, the effects of excess OGF, and a deprivation of OGF-OGFr interaction by an opioid antagonist, naltrexone (NTX), were examined in 3 human cancer cell lines: pancreatic (BxPC-3), colon (HT-29), and head and neck (CAL-27). OGF exposure decreased growth, DNA synthesis, and mitosis, and increased the doubling time from control levels. FACS analysis revealed a marked increase in cells in the G0/G1 phase and compensatory reduction in cells in S and G2/M phases. Consistent with this observation, the percentage of labeled mitosis (PLM) analysis showed a notable increase in the time of the G0/G1 phase. Receptor blockade with NTX increased the rate of growth, length of DNA synthesis and mitotic phases, and decreased doubling time from control values. FACS analysis indicated an increase in the proportion of cells in S and G2/M phases, and a decrease in the number of cells in the G0/G1 phase. PLM evaluation demonstrated a shortening of the length of the S and G2 phases in the 3 cell lines, and decreases in the M and G0/G1 phases in some cancers. These results indicate that OGF action is directed at the G0/G1 phase, but interruption of OGF-OGFr interfacing has widespread repercussions on the cell cycle. The data on blockade of OGF-OGFr during log phase growth suggest a requisite escorting of the growth peptide and its receptor through the cell cycle.

Establishment of a human polyclonal oral epithelial cell line.

To develop a human oral epithelial cell line to constitute a continuous source of cells readily available for human oral epithelial cell research. Oral epithelial cells from a 30-week gestational, stillborn male fetus were grown in serum-free medium and transfected by lipid-mediation with the shuttle vector plasmid, pZ189, containing the T-antigen coding region and replication origin from the SV40 virus. Resulting cultures produced foci of rapidly multiplying cells that failed to senesce, in contrast to controls. The transformed culture, designated GMSM-K, was polyclonal. The original culture possessed a normal human male karyotype, and the transformed line was largely hypotetraploid. Multiple clones, isolated from soft agar studies and low density plating, showed decreased doubling times. Electron microscopy and immunohistochemistry confirmed an epithelial phenotype. Cells did not generate tumors in nude mice. Few human epithelial cell lines are available to investigators and most are tumor-derived. The nontumor-derived GMSM-K line has value as a resource for human oral epithelial cell research.

Mitogenic and oncogenic properties of the small G protein Rap1b.

It has been widely reported that the small GTP-binding protein Rap1 has an anti-Ras and anti-mitogenic activity. Thus, it is generally accepted that a normal physiological role of Rap1 proteins is to antagonize Ras mitogenic signals, presumably by forming nonproductive complexes with proteins that are typically effectors or modulators of Ras. Rap1 is activated by signals that raise intracellular levels of cAMP, a molecule that has long been known to exert both inhibitory and stimulatory effects on cell growth. We have now tested the intriguing hypothesis that Rap1 could have mitogenic effects in systems in which cAMP stimulates cell proliferation. The result of experiments addressing this possibility revealed that Rap1 has full oncogenic potential. Expression of Rap1 in these cells results in a decreased doubling time, an increased saturation density, and an unusual anchorage-dependent morphological transformation. Most significantly, however, Rap1-expressing cells formed tumors when injected into nude mice. Thus, we propose that the view that holds Rap1 as an antimitogenic protein should be restricted and conclude that Rap1 is a conditional oncoprotein.

The Early HPV16 Proteins Can Regulate mRNA Levels of Cell Cycle Genes in Human Cervical Carcinoma Cells by p53-Independent Mechanisms

Cervical carcinoma-associated human papillomavirus type 16 (HPV16) encodes E6 and E7 oncoproteins which inactivate p53 and Rb, respectively, but these interactions are not sufficient to account for the oncogenic potential of the virus. Several viral promoters were shown to be regulated by E6 and E7. To identify genes as cellular targets of the HPV16 early proteins, we transfected a new HPV-negative and p53-mutated cervical carcinoma-derived cell line with either the HPV16 full-length genome or the HPV16 E6 gene. HPV16 clones but not 16E6 clones showed a decreased doubling time that was not related to the viral DNA and mRNA patterns. In exponentially growing cells as well as in cells synchronized by serum starvation, expression of the E6 gene was associated with upregulation of the c-fosand c-junproto-oncogenes and with downregulation of the c-Ha-rasgene. Furthermore, a viral gene other than E6 may be involved in downregulation of p53 because a reduced mRNA level at the G1/S transition was observed only in HPV16-cells. The present study on natural host cells indicates p53-independent transcriptional modulations of cell cycle regulatory genes related to HPV16 E6 and E7 expression.

High susceptibility of an Epstein-Barr virus-converted Burkitt's lymphoma cell line to cytotoxic drugs

Susceptibility to cytotoxic drugs was studied using an Epstein-Barr virus (EBV) genome-negative Burkitt's lymphoma (BL) cell line (Ramos) originating from the tumor cells and an in vitro EBV-converted Ramos cell line (B7). Decreased doubling time (DT) with increased saturation density (SD) and elevated [ 3H]thymidine incorporation were shown in B7, indicating more rapid cell proliferation and growth. However, B7 was highly susceptible to cytotoxic drugs when compared to Ramos. These results indicated whether the presence or absence of EBV genome in tumor cells may be beneficial for evaluation of susceptibility to cytotoxic chemotherapy in patients with BL.

Effect of passage number on cellular response to DNA-damaging agents: cell survival and gene expression.

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or gamma-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60Co gamma rays or 254-nm UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a 'crisis' period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of gamma-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following gamma-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

Mitogenic effects of pertussis toxin-sensitive G-protein α subunits: The mitogenic action of αi2 in NIH 3T3 cells is mimicked by αi1, but not αi3

In fibroblasts and other cell types, pertussis toxin (PTX) inhibits DNA synthesis in response to serum and certain growth factors. GTPase deficient forms of the PTX-sensitive G-protein α i2 subunit have been shown to induce partial transformation in fibroblasts. In order to determine whether other PTX-sensitive G-protein scan stimulate mitogenic pathways, we stably expressed constitutively activated G-protein α i1 and α i3 subunits in NIH 3T3 cells. Expression of activated α i1, α i2 or α i3 results in inhibition of forskolin-stimulated cAMP accumulation in intact cells. Constitutively activated α i1, but not α i3, induces a loss of contact inhibition, a loss of anchorage-dependence, a reduced serum requirement and a decreased doubling time in NIH 3T3 cells. We conclude that α i1 and α i2 are both capable of transducing mitogenic signals, but that α i3 is not involved in the regulation of fibroblast growth. Furthermore, adenylyl cyclase inhibition is clearly not sufficient to explain the effect of α i2 on fibroblast growth.

Autocrine transforming growth factor-alpha is associated with progression of transformed properties in human colon cancer cells.

The GEO colon carcinoma cell line is weakly tumorigenic in athymic mice and shows differentiated properties both in tissue culture and in xenografts. Proliferating monolayer cultures of GEO cells which normally require exogenous epidermal growth factor (EGF) for optimal growth displayed a marked inhibition in growth upon addition of antibodies that block binding to the EGF receptor or neutralize TGF-alpha. These results indicated that GEO cells utilize TGF-alpha in a weak autocrine loop. The availability of a weakly malignant model system in which TGF-alpha had demonstrable, but low level autocrine activity, permitted the investigation of the role of TGF-alpha in tumorigenesis by generating a stronger autocrine loop through the overexpression of the polypeptide. GEO cells were electroporated with an expression vector containing the human TGF-alpha cDNA, and stable clones were isolated that constitutively expressed the TGF-alpha cDNA in a strong autocrine loop. However, the growth rate of the parental cells in EGF-supplemented medium was the same as that of transfected cells with or without growth factor-supplemented medium. Thus, any biological changes generated by the overexpression of TGF-alpha were due to the autocrine nature of the growth mechanism rather than due to any decrease in doubling time leading to a faster growth rate. Transfected GEO cells showed an increase in anchorage-independent growth and formed tumors more readily in athymic nude mice indicating that TGF-alpha plays a role in progression of transformed properties.

Differential effects of the simian virus 40 early genes on mammary epithelial cell growth, morphology, and gene expression

To study the effect of SV40 T-antigen in mammary epithelial cells, a rat β-casein promoter-driven SV40 early-region construct was stably introduced into the clonal mouse mammary epithelial cell line HC11. With the expression of the viral T-antigens under the control of a hormone-inducible promoter, it was possible to dissociate the effects of different levels of T-antigen expression on cell growth, morphology, and gene expression. Following hormonal induction, a rapid but transient induction of T-antigen was observed, followed by a delayed induction of H4 histone mRNA. In T-antigen-positive HC11 cells cultured in the absence of EGF, the expression of basal levels of T-antigen (in the absence of hormonal induction) led to a decreased doubling time and an increased cell density. In the presence of EGF, T-antigen expression resulted additionally in an altered cell morphology. Despite the effects of T-antigen on cell growth and gene expression, the cells were unable to form colonies in soft agar and were nontumorigenic when transplanted into cleared mammary fat pads. They were, however, weakly tumorigenic in nude mice. Relatively high levels of p53 protein synthesis were observed in both the transfected HC11 cells and the parental COMMA-D cells, as compared to 3T3E fibroblasts and another mammary epithelial cell line. The HC11 and COMMA-D cells synthesized approximately equal levels of wild-type and mutated p53 proteins as defined by their reactivities with monoclonal antibodies PAb246 and PAb240, respectively. Interactions between excess p53 and T-antigen may, in part, explain the failure of these cells to display a completely transformed phenotype.

Dimethylformamide-Induced Changes in the Radiation Survival of Low- and High-Passage Intestinal Epithelial Cells (IEC-17) in Vitro

The effects of dimethylformamide (DMF) on the radiation response of low- and high-passage intestinal epithelial cells (IEC-17) were examined. The IEC-17 cell line, a rat intestinal epithelial cell line, exhibited a bimodal response to X radiation. The sensitive fraction, which was attributed to a stem cell-like component, had a D0 of 0.90 Gy. The resistant fraction, thought to be the expression of a more mature component, exhibited a D0 of 2.00 Gy. Treatment using a putative cell differentiating agent, N,N-dimethylformamide, increased the resistant fraction of the population from 35 to 80%, suggesting that DMF treatment (100 mM) increased the proportion of mature cells in the IEC-17 cell population. In addition, extended age in culture (greater than 100 passages) resulted in altered morphology, decreased doubling time, increased chromosome number, and loss of anchorage dependence, all features characterizing spontaneously transformed high-passage IEC-17 cells. These high-passage cells also exhibited a bimodal response to X radiation; the sensitive fraction had a D0 of 0.80 Gy while the resistant fraction D0 was 1.50 Gy. DMF increased the resistant fraction from 35 to 55% of the population. Results suggested that the different radiosensitivities of the subpopulations remained throughout the spontaneous transformation of high-passage IEC-17 cells.

Responsiveness of human carcinoma cells of gynecologic origin to 1,25-dihydroxycholecalciferol

In responsive tissues the induction of 25-hydroxycholecalciferol 24-hydroxylase activity is a marker of 1,25-dihydroxycholecalciferol action. In this study we investigated the responsiveness of various malignant cells, which were derived from carcinomas of gynecologic origin, to 1,25-dihydroxycholecalciferol; we did so because it has been demonstrated that replication of certain malignant human cells that are 1,25-dihydroxycholecalciferol-responsive is inhibited by treatment with this substance. We found that 1,25-dihydroxycholecalciferol treatment caused an increase in 25-hydroxycholecalciferol 24-hydroxylase activity in gynecologic carcinoma cells that were maintained in monolayer culture. The cells were established from ovarian, endometrial, cervical, and vaginal neoplasms. After treatment with 1,25-dihydroxycholecalciferol (10 nmol/L) for 18 hours, the specific activity of 25-hydroxycholecalciferol 24-hydroxylase increased between 2.4- and 49-fold in these cells. This increase in 25-hydroxycholecalciferol 24-hydroxylase activity is suggestive that certain gynecologic malignancies are responsive to 1,25-dihydroxycholecalciferol. We also found, however, that 1,25-dihydroxycholecalciferol did not act to decrease the rate of replication of these cells; indeed in ovarian adenocarcinoma cells, 1,25-dihydroxycholecalciferol caused a decrease in doubling time; in the other cells of this study, there was no effect of 1,25-dihydroxycholecalciferol on cell replication.

Effect of adroxazine, a heterocyclic compound of the adrenals, on the rate of replication of lymphoblastoid cells.

It has been found that the addition of 10(-2)-10(-8) microL/mL of adroxazine, heterocyclic compound of the adrenals, to the tissue culture medium increases the rate at which three strains of lymphoblastoid cells replicate. For example, the addition of 10(-5) micrograms/mL of adroxazine to cultured L5178Y cells decreases their doubling time from approximately 11 to 8 h and addition of 10(-3) micrograms/mL of adroxazine to the tissue culture medium of Huly-16 or Huly-29 cells decreases their doubling time from approximately 43 to 34 and 36 h, respectively. Other concentrations of adroxazine, from 10(-2) to 10(-8) micrograms/mL, cause a less but measurable decrease in doubling time. The effect of adroxazine on the rate of incorporation of [3H]thymidine into acid-precipitable material by lymphoblastoid cells was found to parallel the effect of adroxazine on doubling time. The effect of adroxazine on the length of DNA synthesis and mitosis was calculated from the percent of cells that are labeled with [3H]thymidine during a 20-min interval and the percent of cells in mitosis, respectively. Results showed that adroxazine does not decrease the length of DNA synthesis or mitosis, but markedly decreases the sum of pre- and post-mitotic resting times (TG2 + TG1). Addition of 10(-3) micrograms/mL of adroxazine to the medium decreases the resting times of Huly-16 cells from approximately 20 to 10 h and Huly-29 cells from approximately 12 to 5 h. Addition of 10(-5) micrograms/mL of adroxazine to the medium decreases the resting time of L5178Y cells from 3.3 to 0.1 h.(ABSTRACT TRUNCATED AT 250 WORDS)