Masahiro Hitomi

Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation

Telomerase, the enzyme that elongates telomeric DNA (TTAGGG)n, may be involved in cellular immortality and oncogenesis. To investigate the effect of inhibition of telomerase on tumor cells, we transfected the antisense vector against the human telomerase RNA into human malignant glioma cells exhibiting telomerase activity. After 30 doublings, some subpopulations of transfectants expressed a high level of interleukin‐1β‐converting enzyme (ICE) protein and underwent apoptosis. In contrast, other subpopulations also showed enhanced ICE protein but escaped from apoptotic crisis and continued to grow, although their DNA synthesis, invasive ability, and tumorigenicity in nude mice were significantly reduced. Surviving cells demonstrated increased expression of glial fibrillary acidic protein and decreased motility, consistent with a more differentiated state. These cells also contained enhanced expression of the cyclin‐dependent kinase inhibitors (CDKIs) p21 and p27. Treatment of surviving nonapoptotic cells with antisense oligonucleotides against p27, but not p21, induced apoptotic cell death, suggesting that p27 may have protected differentiating glioma cells from apoptosis. These data show that treatment with antisense telomerase inhibits telomerase activity and subsequently induces either apoptosis or differentiation. Regulation of these two distinct pathways may be dependent on the expression of ICE or CDKIs.—Kondo, S., Tanaka, Y., Kondo, Y., Hitomi, M., Barnett, G. H., Ishizaka, Y., Liu, J., Haqqi, T., Nishiyama, A., Villeponteau, B., Cowell, J. K., Barna, B. P., Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation. FASEB J. 12, 801–811 (1998)

Brain Tumor Initiating Cells Adapt to Restricted Nutrition through Preferential Glucose Uptake

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) owing to preferential BTIC survival and to adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3, and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, tumor initiating cells may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may maintain the tumor hierarchy and portend poor prognosis.

Distribution of CD133 reveals glioma stem cells self-renew through symmetric and asymmetric cell divisions.

Malignant gliomas contain a population of self-renewing tumorigenic stem-like cells; however, it remains unclear how these glioma stem cells (GSCs) self-renew or generate cellular diversity at the single-cell level. Asymmetric cell division is a proposed mechanism to maintain cancer stem cells, yet the modes of cell division that GSCs utilize remain undetermined. Here, we used single-cell analyses to evaluate the cell division behavior of GSCs. Lineage-tracing analysis revealed that the majority of GSCs were generated through expansive symmetric cell division and not through asymmetric cell division. The majority of differentiated progeny was generated through symmetric pro-commitment divisions under expansion conditions and in the absence of growth factors, occurred mainly through asymmetric cell divisions. Mitotic pair analysis detected asymmetric CD133 segregation and not any other GSC marker in a fraction of mitoses, some of which were associated with Numb asymmetry. Under growth factor withdrawal conditions, the proportion of asymmetric CD133 divisions increased, congruent with the increase in asymmetric cell divisions observed in the lineage-tracing studies. Using single-cell-based observation, we provide definitive evidence that GSCs are capable of different modes of cell division and that the generation of cellular diversity occurs mainly through symmetric cell division, not through asymmetric cell division.

Variations in cyclin D1 levels through the cell cycle determine the proliferative fate of a cell.

We present evidence that variations in cyclin D1 levels through the cell cycle are essential for continuing proliferation. Cyclin D1 levels must be high during G1 phase for a cell to initiate DNA synthesis, but then must be suppressed to low levels during S phase to allow for efficient DNA synthesis. This suppression during S phase is apparently regulated by cell cycle position alone and occurs automatically during each cell cycle. If the cell is to continue proliferating, cyclin D1 levels must be induced once again during G2 phase. This induction depends upon the activity of proliferative signaling molecules, and ensures that the extracellular environment continues to be conducive for growth. We propose that the suppression of cyclin D1 levels during each S phase ensures that the subsequent induction during G2 phase, and the resulting commitment to continuing proliferation, is closely linked to the cellular growth environment.

Chemotherapy activates cancer-associated fibroblasts to maintain colorectal cancer-initiating cells by IL-17A

Chemotherapy stimulates cancer-associated fibroblasts to secrete interleukin-17A to provide maintenance cues to support the growth of colorectal cancer-initiating cells.

Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

Phosphorylation of cyclin D1 at Thr 286 during S phase leads to its proteasomal degradation and allows efficient DNA synthesis

Continuing proliferation requires regulation of cyclin D1 levels in each cell cycle phase. Growth factors stimulate high levels during G2 phase, which commits the cell to continue through G1 phase with sufficient cyclin D1 to initiate DNA synthesis. Upon entry into S phase, however, cyclin D1 levels rapidly decline. Our goal is to understand the mechanism and importance of this S-phase suppression. Here, we demonstrate that cyclin D1 levels decline during S phase due to reduced protein stability, without alterations in the rate of protein synthesis. This decline depends upon Thr 286, since mutation of this site eliminates the normal pattern of cyclin D1 suppression during S phase. As evidence that phosphorylation of Thr 286 is responsible for this decline, Thr 286 is shown to be more efficiently phosphorylated during S phase than in other cell cycle periods. Finally, high cyclin D1 levels during S phase are shown to inhibit DNA synthesis. This inhibitory activity presumably blocks the growth of cells with altered cyclin D1 expression characteristics. Abnormal stimulation of cyclin D1 might result in levels high enough to promote G1/S phase transition even in the absence of appropriate growth stimuli. In such cells, however, the levels of cyclin D1 would presumably be too high to be suppressed during S phase, resulting in the inhibition of DNA synthesis.

Platelet-derived growth factor receptors differentially inform intertumoral and intratumoral heterogeneity

Growth factor-mediated proliferation and self-renewal maintain tissue-specific stem cells and are frequently dysregulated in cancers. Platelet-derived growth factor (PDGF) ligands and receptors (PDGFRs) are commonly overexpressed in gliomas and initiate tumors, as proven in genetically engineered models. While PDGFRα alterations inform intertumoral heterogeneity toward a proneural glioblastoma (GBM) subtype, we interrogated the role of PDGFRs in intratumoral GBM heterogeneity. We found that PDGFRα is expressed only in a subset of GBMs, while PDGFRβ is more commonly expressed in tumors but is preferentially expressed by self-renewing tumorigenic GBM stem cells (GSCs). Genetic or pharmacological targeting of PDGFRβ (but not PDGFRα) attenuated GSC self-renewal, survival, tumor growth, and invasion. PDGFRβ inhibition decreased activation of the cancer stem cell signaling node STAT3, while constitutively active STAT3 rescued the loss of GSC self-renewal caused by PDGFRβ targeting. In silico survival analysis demonstrated that PDGFRB informed poor prognosis, while PDGFRA was a positive prognostic factor. Our results may explain mixed clinical responses of anti-PDGFR-based approaches and suggest the need for integration of models of cancer as an organ system into development of cancer therapies.

Post-transcriptional regulation of cyclin D1 expression during G2 phase.

During continuous proliferation, cyclin D1 protein is induced to high levels in a Ras-dependent manner as cells progress from S phase to G2 phase. To understand the mechanism of the Ras-dependent cyclin D1 induction, cyclin D1 mRNA levels were determined by quantitative image analysis following fluorescent in situ hybridization. Although a slight increase in mRNA expression levels was detected during the S/G2 transition, this increase could not explain the more robust induction of cyclin D1 protein levels. This suggested the involvement of post-transcriptional regulation as a mechanism of cyclin D1 protein induction. To directly test this hypothesis, the cyclin D1 transcription rate was determined by run-on assays. The transcription rate of cyclin D1 stayed steady during the synchronous transition from S the G2 phase. We further demonstrated that cyclin D1 protein levels could increase during G2 phase in the absence of new mRNA synthesis. α-Amanitin, a transcription inhibitor, did not suppress cyclin D1 protein elevation as the cells progressed from S to G2 phase, even though the inhibitor was able to completely block cyclin D1 protein induction during reentry into the cell cycle from quiescence. The half life of cyclin D1 protein was shortest during S phase indicating that a change in protein stability might play a role in post-translational induction of cyclin D1 in G2 phase. These data indicate a fundamental difference in the regulation of cyclin D1 production during continuous cell cycle progression and re-initiation of the cell cycle.

Cell cycle arrest and morphological alterations following microinjection of NIH3T3 cells with Puraα

Levels of Purα, a protein implicated in control of both DNA replication and gene transcription, fluctuate during the cell cycle, being lowest in early S phase and highest just after mitosis. Here we have employed a new video time-lapse technique enabling us to determine the cell cycle position of each cell in an asynchronous culture at a given time and to ask whether introduction of Purα protein at specific times can affect cell cycle progression. Approximately 80% of all NIH3T3 cells injected with Purα were inhibited from passing through mitosis. Cells injected with Purα during S or G2 phases were efficiently blocked with a 4N (G2 phase) DNA level, as determined by quantitative DNA photometry of individual cells. Of the cells injected with Purα during G1 phase, 40% experienced a rapid cell death characterized by extreme cellular fragmentation. Of those G1 injected cells which remained viable, approximately equal numbers were arrested with either 2N or 4N DNA levels. Cells arrested by Purα in G2 phase grew to cover a large surface area. These results link fluctuations in Purα levels to aspects of cell cycle control.