Downregulation of estrogen receptor and modulation of growth of breast cancer cell lines mediated by paracrine stromal cell signals.

PURPOSE: Breast cancers have a poorer prognosis if estrogen receptor expression was lost during recurrence. It is unclear whether this conversion is cell autonomous or whether it can be promoted by the microenvironment during cancer dormancy. We explored the ability of marrow-derived stromal cell lines to arrest co-cultured breast cancer cells and suppress estrogen receptor alpha (ER) expression during arrest, facilitating the emergence of estrogen-independent breast cancer clones. METHODS: Cancer cell growth, ER protein, microRNA, and mRNA levels were measured in breast cancer cell lines exposed to conditioned medium from marrow stromal lines in the presence and absence of estrogen and of signaling pathway modulators. RESULTS: We demonstrate that paracrine signaling from the stromal cell line HS5 downregulated ER in T47D and MCF7 breast cancer cells. This occurred at the mRNA level and also through decreased ER protein stability. Additionally, conditioned medium (CM) from HS5 arrested the breast cancer cells in G0/G1 in part through interleukin-1 (IL1) and inhibited cancer cell growth despite the activation of proliferative pathways (Erk and AKT) by the CM. Similar findings were observed for CM from the hFOB 1.19 osteoblastic cell line but not from two other fibroblastic marrow lines, HS27A and KM101. HS5-CM inhibition of MCF7 proliferation could not be restored by exogenous ER, but was restored by the IL1-antagonist IL1RA. In the presence of IL1RA, HS5-CM activation of AKT and Erk enabled the outgrowth of breast cancer cells with suppressed ER that were fulvestrant-resistant and estrogen-independent. CONCLUSIONS: We conclude that marrow-derived stromal cells can destabilize estrogen receptor protein to convert the ER status of growth-arrested ER+ breast cancer cell lines. The balance between stromal pro- and anti-proliferative signals controlled the switch from a dormant phenotype to estrogen-independent cancer cell growth.

mRNA stability control: a clandestine force in normal and malignant hematopoiesis.

This review addresses the scope of influence of mRNA decay on cellular functions and its potential role in normal and malignant hematopoiesis. Evidence is emerging that leukemic oncogenes and hematopoietic cytokines interact with mRNA decay pathways. These pathways can co-regulate functionally related genes through specific motifs in the 3'-untranslated region of targeted transcripts. The steps that link external stimuli to transcript turnover are not fully understood, but include subcellular relocalization or post-transcriptional modification of specific transcript-stabilizing or -destabilizing proteins. Improper functioning of these regulators of mRNA turnover can impede normal cellular differentiation or promote cancers. By delineating how subsets of transcripts decay in synchrony during normal hematopoiesis, it may be possible to determine whether this post-transcriptional regulatory pathway is hijacked in leukemogenesis.

Molecular controls of growth arrest and apoptosis: p53-dependent and independent pathways.

Cell homeostasis is regulated by a balance between proliferation, growth arrest and programmed cell death (apoptosis). Until recently, studies on oncogenesis have focused on the regulation of cell proliferation. The recognition that negative growth control, including growth arrest and programmed cell death, must be understood to comprehend how appropriate cell numbers are maintained and how alterations in any part of the equation can contribute to malignancy has led to a burst of work in this field. This review focuses on what has been learned about distinct settings of negative growth control, analyzing p53-dependent and independent pathways of growth arrest and apoptosis either coupled or uncoupled from differentiation, with an emphasis on the use of hematopoietic cells. The importance of understanding the molecular biology of apoptotic and growth arrest pathways in cancer therapy, and future directions to study negative growth control are addressed as well.

Cell cycle-independent upregulation of p27Kip1 by p21Waf1 in K562 cells.

Cellular differentiation frequently involves sequential peaks in the expression of cyclin-dependent kinase inhibitors (cdki's). For example, an increase in levels of the cdki p27Kip1 follows upregulation of p21Waf1 in several cell types induced to differentiate by diverse stimuli. In this study, we have investigated whether p21Waf1 expression itself, rather than the differentiating agent, could be increasing p27Kip1 protein levels. We used an inducible p21Waf1 expression vector in a K562 leukemic cell model which we had previously shown to initiate differentiation following p21Waf1 upregulation. The current study reports that p21Waf1 upregulated p27Kip1 protein without altering p27Kip1 mRNA levels. This effect did not depend on G1-phase arrest-the increase in p27Kip1 occurred at all phases of the cell cycle. p21Waf1-expressing extracts inhibited phosphorylation of p27Kip1 on threonine-187, leading to decreased ubiquitination and decreased proteasomal destruction of p27Kip1. In K562 cells, upregulation of p27Kip1 by p21Waf1 during differentiation facilitated an ordered transition between these two cdki's, each of which may distinctly influence the differentiation process.

Induction of p21 (WAF-1/CIP1) during differentiation.

The recently cloned protein, p21 (WAF1/CIP1) is a downstream effector of p53, and mediates growth arrest by inhibiting the action of G1 cyclin-dependent kinases. Since cellular differentiation is frequently characterized by G1 arrest, we examined whether p21 upregulation occurs in differentiation. We show that p21 expression is triggered by multiple differentiation-inducing agents in hematopoietic and hepatoma cells through a p53-independent pathway. The dramatic rise in p21 levels occurs as an immediate early response to differentiation inducers. The induction of p21 is coupled to the expression of early differentiation markers, and is uncoupled from apoptosis. Finally, evidence is presented that p21 expression is uncoupled from G1 arrest in the presence of deregulated c-myc.

Suppression of G-CSF-mediated Stat signalling by IL-3.

G-CSF-induced myeloid differentiation of 32Dcl3 murine myeloblast cells is antagonized by concurrent exposure to interleukin-3 (IL-3) or by oncogenic transformation of 32Dcl3 by src- or abl-oncogenes which render the cells IL-3-independent. Recent reports have linked G-CSF-mediated differentiation to the ability of G-CSF to activate Stat3. We hypothesized that IL-3 suppresses 32Dcl3 differentiation in part through disruption of G-CSF-Stat signalling. We report that IL-3 inhibited the ability of G-CSF to induce Stat3 DNA binding. Moreover, we find that G-CSF activation of Stat3 binding to DNA is biphasic, peaking at 15-30 min and again at 6-8 h; both peaks are inhibited by IL-3. Transformation of 32Dcl3 cells by the v-abl oncogene leads to constitutive Stat3 activation and distinctive Stat-DNA-binding patterns which are not affected by G-CSF. Cross-modulation of Stat pathway signalling could be a physiologic mechanism for establishing a hierarchy of growth factor effects upon a cell exposed at once to multiple cytokines.

Clonal response of K562 leukemic cells to exogenous p21WAF1.

The p21WAF1 protein is involved in the control of cell differentiation and proliferation. We have previously shown that p21WAF1 is upregulated in normal, proliferating hematopoietic cells undergoing differentiation. Exogenous p21WAF1 has been reported to increase colony-formation by normal hematopoietic progenitors. We examined the effects of exogenous p21WAF1 on proliferation, differentiation, gene expression and colony-formation by K562 cells using an inducible p21WAF1 expression construct. Expression of the stathmin (oncoprotein 18) gene decreased within 24 h of p21WAF1 expression; Hox B4 expression increased. Four K562 subclones were derived which differed in their response to equivalent induction of p21WAF1. All four subclones exhibited growth arrest in response to p21WAF1 in liquid culture. Three of four clones developed cytoplasmic granulation and partial morphologic differentiation after p21WAF1 induction. One clone exhibited fewer morphologic features of differentiation following p21WAF1 induction and unlike other clones, colony formation in methlycellulose was not decreased by p21WAF1 expression in this clone. This indicates that additional cell-specific factors influence cellular fate in the presence of elevated p21WAF1.

p21WAF1 prevents down-modulation of the apoptotic inhibitor protein c-IAP1 and inhibits leukemic apoptosis.

BACKGROUND: Prior studies indicate that leukemias expressing high levels of the p21WAF1 cell cycle inhibitor have a poorer prognosis than p21WAF1-negative leukemias. Although p21WAF1 is upregulated by p53 in the setting of DNA damage, the prognostic significance of p21WAF1 is independent of p53 status. The molecular basis of the negative prognostic effect of p21WAF1 remains obscure, but it is believed to result from decreased apoptosis of p21WAF1-expressing leukemias. MATERIALS AND METHODS: We studied the effects of p21WAF1 on apoptosis of K562 leukemic cells, which lack wild-type p53 and do not express endogenous p21WAF1. An inducible p21WAF1 system was used and the effect of p21WAF1 induction on susceptibility to etoposide-mediated apoptosis was measured. RESULTS: p21WAF1 decreased apoptotic death of K562 leukemic cells in response to etoposide. Analysis of intermediaries in the apoptotic pathway indicated that p2 WAF1 had no effect on cytochrome c release or cleavage of procaspase-3. In contrast, p21WAF1 was protective against cleavage of caspase targets poly(ADP-ribose)polymerase (PARP), retinoblastoma protein (Rb), and lamin. The expression of the inhibitor of apoptosis protein c-IAP1, which inhibited the function of executioner caspases 3 and 7, was studied. c-IAP1 protein expression was found to be present in a majority of leukemic blasts from untreated patients, but absent in normal differentiating myeloid progenitor cells. In K562 cells, treatment with etoposide in the absence of p21WAF1 induction resulted in post-transcriptional down-modulation of c-IAP1 levels. c-IAP1 loss involved proteasomal, rather than caspase, degradation pathways. Expression of p21WAF1 sustained c-IAP1 protein levels in the presence of etoposide. CONCLUSIONS: Etoposide-mediated apoptosis involves down-modulation of the anti-apoptotic protein c-IAP1. Our findings support the hypothesis that p21WAF1 contributes to leukemic chemoresistance by stabilizing c-IAP1 levels in the presence of chemotherapy.

Granulocyte colony-stimulating factor receptor mRNA upregulation is an immediate early marker of myeloid differentiation and exhibits dysfunctional regulation in leukemic cells.

The identification of early markers of myeloid differentiation can facilitate an understanding of how differentiation is arrested in leukemogenesis. Using murine bone marrow and the granulocyte-precusor cell line 32Dc13, we show that message for the granulocyte colony-stimulating factor receptor (G-CSFR) is upregulated by G-CSF in an immediate early fashion that is specific to the differentiation pathway and is antagonized by interleukin-3. We further show that G-CSFR message is superinduced by cycloheximide and that these patterns of regulation are altered in leukemic cell lines. In particular, the v-abl oncogene product supresses both ligand-mediated upregulation and superinduction of the G-CSFR gene.

Differential regulation of transcription of p21 and cyclin D1 conferred by TAF(II)250.

The TATA-binding protein-associated factor TAF(II)250, the largest subunit of TFIID, was first identified as the cell cycle regulatory protein CCG1. The ts13 Syrian hamster ovary fibroblast cell line, which contains a temperature-sensitive point mutation in TAF(II)250/CCG1, is arrested in G1 following a shift to the nonpermissive temperature. Here we demonstrate that the level of the D-type cyclins, in particular D1, was reduced, whereas the level of the cyclin-dependent kinase inhibitor p21 was stimulated in ts13 cells at the nonpermissive temperature. The levels of expression of cyclins A and E were not affected by temperature shift. We further show that at least part of the regulation of D1 and p21 levels in ts13 cells is mediated at the level of transcription initiation. These results suggest that the effect of the temperature-sensitive mutation TAF(II)250 on cell growth can be mediated through the differential regulation of transcription of specific cell growth regulatory genes, such as cyclin D1 and p21.

An element of the BK virus enhancer required for DNA replication.

The human papovavirus BK virus contains three 68-base-pair (bp) repeats that act as transcriptional enhancers. An analysis of plasmids containing the BK virus origin revealed that sequences within the 68-bp enhancer are required for DNA replication as well as transcription of the early promoter in COS-1 cells. Origins with a single 68-bp repeat replicated as efficiently as did those with three repeats when transfected into COS-1 cells. Replication did not occur in the absence of enhancer sequences and could not be restored by distal placement of enhancers to enhancerless origins. However, as with simian virus 40, replication in vitro was not dependent on the presence of any enhancer sequences. Deletion analysis showed that replication of BK virus origins was dependent on the presence of the first 21 bp of the enhancer contiguous with the A-T-rich stretch of the origin. This 21-bp element is referred to as the rep element. Although in combination with rep the remaining 47 bp of the enhancer appear to increase replication by two- to fivefold, they alone are not sufficient to support replication. Deletions or insertions in the enhancer which did not alter the rep element had no major effect on replication. Site-directed mutagenesis of the Sp1-like site within the rep element, the NF1 site present in the enhancer, or the NF1 site in adjacent late-side sequences each reduced transcription by two- to fivefold, but had no effect on replication, suggesting that replication and transcription can be uncoupled.

Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells.

A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34(+) blast cells along the myeloid lineage. We find p27 but not p21 to be expressed in freshly harvested resting CD34(+) cells. Thereafter, p21 levels peak concurrent with cellular proliferation and then decline in expression as cells undergo terminal differentiation. In contrast, p27 levels are fairly constant but the subcellular localization of p27 changes from nuclear expression to predominantly cytoplasmic expression and finally to perinuclear localization at progressive stages of differentiation. This report discusses the implications of these findings.

Dynamic changes in p27kip1 variant expression in activated lymphocytes.

The p27Kip1 cell cycle inhibitor (p27) has emerged as a critical mediator of normal cellular growth control. We report the expression of a 24 kD C-terminal variant of p27 in normal peripheral blood lymphocytes. This variant is rapidly degraded in a proteasome-dependent manner when lymphocytes are activated by interleukin-2 or by superantigen. Whereas p24 degradation is complete within 16 h of mitogen addition, full-length p27 is decreased only modestly over 72 h of mitogen exposure and is present in activated and cycling lymphocytes. Persistent p27 is present in a complex with cyclin D3 in activated lymphocytes, and is localized both in the nucleus and cytoplasm. These results indicate that lymphocytes exiting from quiescence use several mechanisms to overcome the p27Kip1-enforced cell cycle checkpoint, and that elimination of p27 is not required for cell cycle entry.

Molecular cloning of cDNAs for the human granulocyte colony-stimulating factor receptor from HL-60 and mapping of the gene to chromosome region 1p32-34.

Early studies examining the effects of purified or recombinant granulocyte colony-stimulating factor (G-CSF) on human leukemia cell lines demonstrated that some cell lines, such as HL-60, could be induced to differentiate in response to G-CSF. In two recent studies reporting the cloning of the human G-CSF receptor (hGCSFR), four classes of receptor cDNA were identified and, surprisingly, the message for this receptor was reportedly expressed by HL-60 at either very low levels or not at all. Using a mouse G-CSF receptor probe, we cloned and sequenced a cDNA for hGCSFR from an HL-60 cDNA library in plasmid and used it to identify 31 additional clones from an HL-60 cDNA library in phage. Polymerase chain reaction analysis of the 31 phage clones established that 29 were derived from class I hGCSFR mRNA, one was derived from class III mRNA, and one was derived from class IV mRNA. In addition, the hGCSFR gene was chromosomally localized by Southern blot analysis of its segregation pattern in a panel of rodent-human hybrid DNAs using the radiolabeled cDNA probe. The hGCSFR locus was present in hybrids retaining the distal short arm of human chromosome 1 and absent in hybrids that did not retain this region. Chromosomal in situ hybridization refined the localization of the hGCSFR gene to region 1p32-p34. The combination of hybrid DNA analysis and in situ hybridization places the hGCSFR gene telomeric to the CSF1, JUN, and TCL-5 loci.

Regulation of p21(WAF1) expression during normal myeloid differentiation.

The G1-phase cell-cycle inhibitor p21 has been proposed to mediate growth arrest during differentiation. Upregulation of p21 has been shown in multiple cell lines induced to differentiate; however, the mechanism of p21 induction during normal differentiation is largely unknown. In this report, we use normal hematopoietic precursor cells obtained from umbilical cord to model p21 regulation during differentiation. Myeloid maturation of CD34+ precursor cells is associated with a marked increase in p21 expression at the RNA and protein level. The upregulation of p21 transcripts during differentiation is associated with decreased binding to a highly conserved 44-bp fragment within the p21 promoter. This 44-bp regulatory element binds a novel modulator of p21 expression. It is of considerable interest that, although the binding activity is expressed in p53-negative as well as in p53-positive cells, the DNA sequence recognized by this protein overlaps a PuPuPuC(A/T)(T/A)GPyPyPy consensus sequence for p53.

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling.