Apolipoprotein E inhibits serum-stimulated cell proliferation and enhances serum-independent cell proliferation

Independently of its role in lipid homeostasis, apolipoprotein E (apoE) inhibits cell proliferation. We compared the effects of apoE added to media (exogenous apoE) with the effects of stably expressed apoE (endogenous apoE) on cell proliferation. Exogenous and endogenous apoE increased population doubling times by 30-50% over a period of 14 days by prolonging the G(1) phase of the cell cycle. Exogenous and endogenous apoE also decreased serum-stimulated DNA synthesis by 30-50%. However, apoE did not cause cell cycle arrest; both apoE-treated and control cells achieved equivalent saturation densities at 14 days. Further analyses demonstrated that exogenous and endogenous apoE prevented activation of MAPK but not induction of c-fos expression in response to serum growth factors. Endogenous (but not exogenous) apoE altered serum concentration-dependent effects on proliferation. Whereas control (non-apoE-expressing) cell numbers increased with increasing serum concentrations (1.6-fold for every 2-fold increase in serum), apoE-expressing cell numbers did not differ as serum levels were raised from 2.5 to 10%. In addition, in low serum (0.1%), apoE-expressing cells had elevated DNA synthesis levels compared with control cells. We conclude that apoE does not simply inhibit cell proliferation; rather, the presence of apoE alters the response to and requirement for serum mitogens.     

Gonadal dimorphism explained as a dosage effect of a locus on the sex chromosomes, the gonad-differentiation locus (GDL).

In human somatic cells bearing two X chromosomes, one X is genetically inactivated throughout most of its length, whereas in cells with one X and one Y both sex chromosomes are active (with the exception of the constitutive heterochromatin of the Y that is inert). The vast base of information concerning normal and abnormal human sexual development that has accumulated since the advent of human cytogenetics 3 decades ago can be integrated by the following hypothesis: Homologous gonad-differentiation loci (GDLs) exist on the X and Y. The GDLs are strictly sex-linked; that is, normally they do not recombine during spermatogenesis, so that considerable divergence in DNA sequence doubtless has occurred between the locus on the X and the locus on the Y. The abundance of their evolutionarily conserved product--a substance still to be identified--determines the path of differentiation that the indifferent gonadal anlage of the early embryo will take: if only one GDL is transcribed, the case when two X chromosomes are present, ovary will develop; if two GDLs are transcribed, the case when a Y is present along with an X, testis will develop. By implication, facultative X inactivation is an integral and essential component of the system adopted in mammalian evolution for accomplishing gonadal--viz., sexual--dimorphism.     

Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

Oncogenes and cell proliferation

The control of cell proliferation has an impact on both basic and practical problems in biology. Many of these problems intersect in the study of cancer biology, which is one organizing focus of this issue of Current Opinion in Genetics & Development.     

Canine dimorphism

Canine dimorphism in many primates is exaggerated, with males possessing enormous, sharp canines that project far beyond the occlusal plane of the other teeth and females having smaller, less projecting canines. Ever since Darwin,¹ canine dimorphism generally has been attributed to sexual selection. However, recent analyses suggest that the evolution of canine dimorphism is complex and that the sexual selection hypothesis is only part of the story.     

Calcium signalling and cell proliferation

The orderly sequence of events that constitutes the cell cycle is carefully regulated. A part of this regulation depends upon the ubiquitous calcium signalling system. Many growth factors utilize the messenger inositol trisphosphate (InsP₃) to set up prolonged calcium signals, often organized in an oscillatory pattern. These repetitive calcium spikes require both the entry of external calcium and its release from internal stores. One function of this calcium signal is to activate the immediate early genes responsible for inducing resting cells (G₀) to re-enter the cell cycle. It may also promote the initiation of DNA synthesis at the G₁/S transition. Finally, calcium contributes to the completion of the cell cycle by stimulating events at mitosis. The role of calcium in cell proliferation is highlighted by the increasing number of anticancer therapies and immunosuppressant drugs directed towards this calcium signalling pathway.     

Parity models of cell proliferation

Models of the cell cycle are developed and simulated for renewing and for exponentially growing populations. Type A corresponds to the author's previous model (Barrett, 1966, Barrett, 1966). Type B incorporates a resting or regenerating phase, recurring regularly every few cycles, with an endogeneous rhythm. The parity of a cell is the number of cycles since the phase occurred in an ancestral cycle. Type C incorporates asymmetric cell division. Type B leads to novel features in calculated and simulated labelled mitoses curves, since increasing amplitudes of successive waves are permitted, and accords with certain tumour data that previously seemed anomalous. Type B leads also to correlations between cycle times for different generations, in accordance with patterns reported in bacteria. Some mathematical properties of models Type A and B are noted.     

Normal versus abnormal cell proliferation

The most recent findings on the molecular and cellular characterization of normal and abnormal cell proliferation are summarized. They include molecular spectroscopy, nucleic acid conformation, protein modifications, premature chromosome condensation, nuceoli changes, nuclear and cell morphometry, image analysis, flow microfluorimetry, and time-lapse cinematography. Biophysical and biochemical evidence in favor or against two cycles of chromatin condensation, followed by two abrupt random decondensations, per cell cycle are presented. Other biphasic changes at the molecular and cellular levels that favor the existence of two random transitions, or restriction points, per cell cycle are discussed. A comprehensive unitary model of the cell cycle is then outlined; this model is able to explain most findings on continuously dividing cells and on quiescent cells induced to proliferate. Within this analytical framework the physical-chemical and biological properties are given, in either normal or tumor cells, for the various types of “noncycling” cells that are here viewed as necessary steps in mammalian cell growth rather than separate states. The implications of the coupling of higher-order chromatin structure with cell geometry and growth, high in fibroblast-like cells but low in transformed cells, are also discussed. Molecular mechanisms likely responsible for the chromatin conformational changes occurring at the G₀→G₁, G₁→S, G₂→M transitions are finally discussed in terms of polyelectrolyte theory.     

Automated topographical cell proliferation analysis

BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled anti-BrdU and counterstained with To-Pro-3 are scanned using confocal laser scanning microscopy (CLSM). For every point in the image, the nucleus density of BrdU-labeled nuclei and the total nucleus density of the neighborhood of that point are calculated from the BrdU and the To-Pro-3 signal, respectively. The ratio of these densities gives an indication of the amount of cell proliferation at that point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manual count. RESULTS: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calculating the topographical cell proliferation using the automated method is faster and does not suffer from interobserver variability. CONCLUSIONS: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissue. This can be visualized by a topographical map that corresponds to the tissue under study.     

Aldehyde dehydrogenases and cell proliferation

Aldehyde dehydrogenases (ALDHs) oxidize aldehydes to the corresponding carboxylic acids using either NAD or NADP as a coenzyme. Aldehydes are highly reactive aliphatic or aromatic molecules that play an important role in numerous physiological, pathological, and pharmacological processes. ALDHs have been discovered in practically all organisms and there are multiple isoforms, with multiple subcellular localizations. More than 160 ALDH cDNAs or genes have been isolated and sequenced to date from various sources, including bacteria, yeast, fungi, plants, and animals. The eukaryote ALDH genes can be subdivided into several families; the human genome contains 19 known ALDH genes, as well as many pseudogenes. Noteworthy is the fact that elevated activity of various ALDHs, namely ALDH1A2, ALDH1A3, ALDH1A7, ALDH2*2, ALDH3A1, ALDH4A1, ALDH5A1, ALDH6, and ALDH9A1, has been observed in normal and cancer stem cells. Consequently, ALDHs not only may be considered markers of these cells, but also may well play a functional role in terms of self-protection, differentiation, and/or expansion of stem cell populations. The ALDH3 family includes enzymes able to oxidize medium-chain aliphatic and aromatic aldehydes, such as peroxidic and fatty aldehydes. Moreover, these enzymes also have noncatalytic functions, including antioxidant functions and some structural roles. The gene of the cytosolic form, ALDH3A1, is localized on chromosome 17 in human beings and on the 11th and 10th chromosome in the mouse and rat, respectively. ALDH3A1 belongs to the phase II group of drug-metabolizing enzymes and is highly expressed in the stomach, lung, keratinocytes, and cornea, but poorly, if at all, in normal liver. Cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons or chlorinated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, in rat liver cells and increases during carcinogenesis. It has been observed that this increased activity is directly correlated with the degree of deviation in hepatoma and lung cancer cell lines, as is the case in chemically induced hepatoma in rats. High ALDH3A1 expression and activity have been correlated with cell proliferation, resistance against aldehydes derived from lipid peroxidation, and resistance against drug toxicity, such as oxazaphosphorines. Indeed, cells with a high ALDH3A1 content are more resistant to the cytostatic and cytotoxic effects of lipidic aldehydes than are those with a low content. A reduction in cell proliferation can be observed when the enzyme is directly inhibited by the administration of synthetic specific inhibitors, antisense oligonucleotides, or siRNA or indirectly inhibited by the induction of peroxisome proliferator-activated receptor γ (PPARγ) with polyunsaturated fatty acids or PPARγ transfection. Conversely, cell proliferation is stimulated by the activation of ALDH3A1, whether by inhibiting PPARγ with a specific antagonist, antisense oligonucleotides, siRNA, or a medical device (i.e., composite polypropylene prosthesis for hernia repair) used to induce cell proliferation. To date, the mechanisms underlying the effects of ALDHs on cell proliferation are not yet fully clear. A likely hypothesis is that the regulatory effect is mediated by the catabolism of some endogenous substrates deriving from normal cell metabolism, such as 4-hydroxynonenal, which have the capacity to either stimulate or inhibit the expression of genes involved in regulating proliferation.