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Effects of Biofield Therapy on Calcium Release in Immortalized Mouse Keratinocyte HaCaT Cells

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  • Secretaria Nacional De Educación Superior, Ciencia, Tecnología E Innovación Del Ecuador
Article

Effects of Biofield Therapy on Calcium Release in Immortalized Mouse Keratinocyte HaCaT Cells

Abstract and Figures

Objective: Observe the response on the release of calcium into mouse keratinocyte HaCaT cells subjected to biofield therapy (BT) through a pranic healing technique. Design: This was a pilot experimental study. Settings/location: The study was conducted in a laboratory at Simon Bolivar University. Subjects: Mouse keratinocyte HaCaT cells. Interventions: The intervention consisted of a 15-minute biofield therapy using a pranic healing technique. Outcome measures: Cells were loaded with 5 μM calcium indicator Fura 2-AM to monitor changes in intracellular calcium concentration. Cell population was separated into two groups: a control group where cells received no stimulation and the other experimental group where pranic healing was applied. Results: The cells that were treated with pranic healing showed a significant increase in intracellular calcium concentration as compared with untreated cells. Such increase in calcium concentration is consistent with the depletion of intracellular stores. By the action of Thapsigargin (TG) peak, calcium release is equivalent to cells exposed to pranic healing in comparison to control cells. One possible explanation for this observed result is that store of intracellular calcium had been stimulated by pranic healing and hence the resulting drain is lower. These results allow us to infer that pranic healing has an action on intracellular calcium storage but does not allow us to clarify how calcium has been stimulated.
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DOI: 10.5963/PHF0402002
Effects of Biofield Therapy on Calcium Release in
Immortalized Mouse Keratinocyte HaCaT Cells
Silva Ricardo*1, Herrera Alvaro2, Velásquez Hordep3
1Prometeo Program, Coordinación Zonal 5 y 8, Senescyt, Av. Carlos Luis Plaza Dañín, Edificio del Sector Público del Sector
Social, quinto piso, Guayaquil, Ecuador
1Facultad de Ciencias Médicas, Universidad de Guayaquil, Cdla. Universitaria Salvador Allende Malecón del Salado,
Guayaquil, Ecuador
1,2,3Laboratorio de Biociencias Integradas, Universidad Simon Bolivar, Sartenejas, Baruta, Edo. Miranda - Apartado 89000
Cable Unibolivar, Caracas, Venezuela
*1ricardo.silvab@ug.edu.ec
Abstract-Objective: Observe the response on the release of calcium into mouse keratinocyte HaCaT cells subjected to biofield
therapy (BT) through a pranic healing technique. Design: This was a pilot experimental study. Settings/location: The study was
conducted in a laboratory at Simon Bolivar University. Subjects: Mouse keratinocyte HaCaT cells. Interventions: The intervention
consisted of a 15-minute biofield therapy using a pranic healing technique. Outcome measures: Cells were loaded with 5 μM calcium
indicator Fura 2-AM to monitor changes in intracellular calcium concentration. Cell population was separated into two groups: a
control group where cells received no stimulation and the other experimental group where pranic healing was applied. Results: The
cells that were treated with pranic healing showed a significant increase in intracellular calcium concentration as compared with
untreated cells. Such increase in calcium concentration is consistent with the depletion of intracellular stores. By the action of
Thapsigargin (TG) peak, calcium release is equivalent to cells exposed to pranic healing in comparison to control cells. One possible
explanation for this observed result is that store of intracellular calcium had been stimulated by pranic healing and hence the
resulting drain is lower. These results allow us to infer that pranic healing has an action on intracellular calcium storage but does not
allow us to clarify how calcium has been stimulated.
Keywords- Fura-2 AM; Mouse Keratinocyte HaCaT Cells; [Ca]i; Issuing Biofield Therapy (BT); Pranic Healing Technique;
Information Theory; Biofield
I. INTRODUCTION
It is difficult to define complementary and alternative medicine (CAM, for short), because this is a very broad field
constantly evolving. The terms of complementary/alternative/nonconventional medicine are used interchangeably, to define a
set of practices that do not meet the requirements of “main stream” evidence based medicine. The World Health Organization
(WHO) considers traditional medicine as: the sum of knowledge, skills and practices based on the theories, beliefs and
experiences indigenous to different cultures (whether explicable or not), used in the maintenance of health, as well as in the
prevention, diagnosis, improvement or treatment of physical and mental illnesses [1]. Some countries around the world have
established institutions and governmental agencies in order to address these issues. The best example is the United States of
America with the creation and operation of the National Centre for Complementary and Alternative Medicine (NCCAM) [2],
which is a dependency of the National Institutes of Health (NIH). According to the NCCAM Complementary, alternative
medicine is defined as a set of systems, practices and products that, in general, not considered a part of conventional medicine.
NCCAM considers Energy Based Medicine as a kind of therapeutic approach which involves the manipulation of various
energy fields that affect the health. Practices based on “assumptions” of energy fields, also called biological fields (biofield),
usually reflect the concept that human beings are engaged in subtle forms of energy [1, 2]. It includes procedures based on
external bioenergy emission (EBE), which brings the things together: Qi gong, therapeutic touch, reiki, pranic healing and
other techniques. The existence of these fields has not yet been scientifically proven, thus the basic science to give support for
such practices is needed. In this article, we propose a biological model that could be used as a biosensor to prove and test such
biofields.
One of the difficulties presented by the study of the therapeutic approaches of CAM and in particular the energy-based
medicine has been the action of the placebo effect and the effect of suggestion on the individual. Therefore studies on Biofield
Therapies (BT), such as Pranic Healing (PH), must apply the same standards to using in the design of experiments in physics,
chemistry and other scientific disciplines focusing on the use of biomodels, such as cells, tissues and experimental animals [1,
2].
Energy Based Medicine posits that a human’s physical body is really composed of two parts: the visible physical body and
the invisible energy body. The visible physical body is the part we see and touch, the energy body is invisible, interpenetrating
the visible physical body and extending beyond this [3]. The human being is a system of energies that are in constant vibration,
i.e., molecules that are composed of atoms and their subatomic particles are in constant motion and interaction. For our
purposes, this energy designated as prana, ki, qi, life force, pneuma, mana, ruach, “breath of life” among other terms, will be
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DOI: 10.5963/PHF0402002
designated with the Greek letter χ (Xi capital letter).
Pranic Healing is an ancient science of healing, using χ to heal the whole physical body. Pranic Healing is based on two
laws: the law of self-healing and the law of prana (or vital energy) [3].
The law of self-healing states that the body is able, to some extent, to heal. If a person has a wound or burn, the body
will heal and recover.
The vital power law states that the body must have χ for life to exist. The healing process can be accelerated by
increasing the amount of χ in the parties’ concern.
One of the major obstacles scientific researchers faced with on alternative medical practices is the placebo controversy.
According to Fabrizio Benedetti and colleagues [4]: “the placebo effect is a psychobiological phenomenon that can be
attributable to different mechanisms, including the expectation of clinical improvement and pavlovian conditioning”.
According to [4], “subjective” constructs such as expectation and value have identifiable physiological bases, and these bases
are powerful modulators of perceptual, motor, and internal homeostatic processes. The physiological bases of the placebo
effect have been supported by multiple authors [5-8]. Without discussion of the inherent physiological pathways for placebo,
the underlying mechanism of operation can be summarized as follows: An inducer or motivator (substance, device, expectation,
conditioning, ritual, etc.), will interact with the mind of a patient, producing a “subjective construct” (SC). SC will trigger a
neurohormonal response, producing a physiological outcome (Fig. 1).
Fig. 1 Effects of cognitive expectations on neural chemical functions and physiological response (placebo effect)
As seen from Fig. 1, all of the traditional medical practices and CAM could fit in the category of motivators. In the case of
traditional evidence based medicine, there is a general consensus on the fact that “medicine” interacts directly with the
metabolism, producing a physiological outcome; therefore, the “motivational” aspect of medicine falls onto a second plane.
The case of “Energy Based Medicine” and BT, is critical, since the sole element of therapy is a practitioner who claims that
he/she can “project” a form of energy that will interact with the patient and produce a response. It is assumed that BT is purely
“motivational” or nothing more than placebo. This is what we define as the Placebo Controversy, In order to break apart from
this controversy; we need to strip BT from “motivational effects”, in a sense, we need to strip them from mind/body
interactions.
Scientists have made several approximations [9, 10] to explain the concept of biofield and energy projection. The focus on
BT raises interesting challenges to the scientific community to try to find relationships between the responses elicited in
biological structures as an effect of stimulus from BT. Cellular studies with immortalized cell lines of T-lymphocyte culture
show that BT application increases intracellular calcium [11], and that this fact is not related to the activation of heat shock
proteins [12]. These experiments are directed towards assessing cause and effect, but the means of this cause effect relation is
yet to be understood.
II. MATERIALS AND METHODS
For this study, we utilized immortalized mouse keratinocyte HaCaT cells [13]. HaCaT is a spontaneously immortalized
human keratinocyte cell line, which develops through long-term culture of normal human adult skin keratinocytes at reduced
calcium concentration and elevated temperature [14].
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A. [Ca2+]i Determinations
For the [Ca2+]i measurements, the fluorescent ratiometric Ca2+ indicator Fura 2 was used, because its excitation spectrum
depends on the concentration of the cation, while its emission peak remains invariable.
The cells were cultured in a defined culture medium containing calcium-free DMEM, 9% FBS and EGF [15]. The cells
were seeded on tissue culture plastic and expanded using standard conditions for a period of two weeks. Cells showed the
typical basal keratinocyte morphology. After expansion, the cells were detached by trypsinization. Isolated cells were
incubated in a solution containing the following composition: 20 mM Hepes, 120 mm NaCl, 5 mM KCl, 1 mM MgCl, 1.5 mm
CaCl2, 1 mg / ml glucose and sodium pyruvate. pH: 7.4. Excess solution was removed and cells were incubated with 5 µM of
a fluorescent indicator, Fura 2- AM [16] in 400 ml of the same buffer for 45 min in darkness and with constant stirring. Cells
were centrifuged and placed in 500 ml of the same buffer and transferred into cuvettes for the measurement [17, 18].
For the experimental setup, two cuvettes were used: a control, where cells were not stimulated; and a test cuvette, where an
experienced PH practitioner projected vital energy (VE) into it. The procedure used to energize the cells is called the palms
technique, where the practitioner has the intention to absorb VE with one hand, and also has the intention of projecting this
energy with the other [3]. In this case, the practitioner’s right hand was at an approximate distance of 10 cm from the test
cuvette for a period of 15 min. As reported by the PH expert, his intention was to energize the cells inside the cuvette.
After this, the cuvette was placed for measurement of fluorescence in a fluorescence spectrometer Perkin Elmer LS-55
provided with an acquisition system for excitation ratio measurements at 29°C with continuous agitation in a stirred cuvette.
[Ca2+]i were evaluated by applying the equation [Ca2+]i = Kd × [(R-Rmin)/(Rmax-R)] × Fmin/Fmax, as reported by [16]; R is
the fluorescent emission ratio obtained after excitation at 340 nm/380 nm; Rmax and Fmax are the ratios of excitation
fluorescence at 340 nm/380 nm and the fluorescence of Fura 2 at 380 nm, respectively, under saturated Ca2+ concentrations;
and Rmin and Fmin are the ratios of excitation fluorescence at 340 nm/380 nm and the fluorescence of Fura 2 at 380 nm,
respectively, in the absence of Ca2+. Maximum and minimum values were obtained after the addition of 30 μM Thapsigargin
(TG). TG is a lactone, non-competitive inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pumps. TG raises
intracellular calcium concentration by blocking the ability of the cell to pump calcium into the sarcoplasmic and endoplasmic
reticula.
The control cuvette remained under the same conditions of temperature, pressure and light, at the same period of time (15
min), a student placed his hand about 10 cm above the cells, with the goal of “shading” the cells from the light. Shading from
the light was the excuse used by the researchers, in order to replicate conditions similar to experimental conditions. Fig. 2
presents a schematic of the experimental setup.
Fig. 2 Schematic of the experimental setup: Tripsinized HaCat cells loaded with Fura 2-AM (1) were placed into two cuvettes (2, 3). VE (4) was projected
onto test cuvette (2). Both cuvettes (2, 3) where placed in a fluorescence spectrometer Perkin Elmer LS-55 (5). Fluorescent measurement results for test (6)
and control (7). The black arrow indicates the application of Thapsigargin (TG) for both samples
The study was double blinded. Cell culture and cell preparation for fluorescent measurements were performed by a
graduate student in cell biology who was not aware of the methods and procedures of BT or PH techniques. Once the cuvettes
were prepared, they were handled by the authors, with the participation of the PH practitioner, and submitted to a fluorescent
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technician for measurement. The technician was also not aware of the performed procedures.
III. RESULTS
Fig. 3 shows two superimposed curves, corresponding to one test and one control cuvette. Cells were loaded with 5 µM
calcium indicator Fura 2-AM to monitor changes in the concentration of intracellular calcium. Cells that have not been
energized (control cells, B) produced lower fluorescence intensity compared with cells that have previously been energized
(test cells, A), before the application of TG.
Fig. 3 Fluorescent measures for HaCaT cells loaded with Fura 2-AM. Control cells (B) and cells treated with BT (A). The black arrow indicates the application
of Thapsigargin (TG)
Note that the fluorescence intensity of control cells is stable up to the point where TG is applied. With the application of
TG, an increase of fluorescence intensity is observed. The quantity of intracellular calcium was computed by using the
conversion factors found in [18] with kd= 224 nm. Fig. 4 shows the result of four experiments. Three points are compared: The
amount of intracellular Ca at the beginning of the measurement, the amount of Ca after 400 ms, and the maximum amount of
Ca2+ after TG application. At 400 ms, the amount of Ca2+ for the control group is 380,80 ± 18,29 nm while test group is 694,40
± 70,84 nm (R2 1,4063E-124), the mean slope (400 ms window) for control group is 0,1260 ± 0,0705 nm/ms while test group
is 0,7000 ± 0,0970 nm/ms.
Fig. 4 Comparison of mean Ca2+ concentration at three points of the fluorescent measurements for four consecutive experiments: at the beginning of the
experiment, 400 ms later and the maximum amount of Ca after TG application
IV. DISCUSSION
In this study, the intracellular calcium concentration [Ca2+] for immortalized mouse keratinocyte HaCaT cells was
monitored with the fluorescent indicator Fura 2-AM. Changes in fluorescence intensity are proportional to calcium fluctuations
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and can be computed using an intensity conversion factor. Cells exposed to VE showed higher basal calcium concentration
compared with control cells (untreated cells). By including the student hand in the vicinity of the cells, it can be shown that the
human hand is not enough to elucidate the response and that some type of stimulus is provided by the PH Practitioner. Calcium
concentration increased steadily in cells treated with BT (approximately 0.7 nm/ms) as compared with control cells which
remained steady until TG addition.
Both the extracellular Ca+2 and the one located within intracellular deposits, can contribute to increase in cytosolic
concentration of the cation in response to external stimuli such as VE. Although the objective of this study was not to
characterize the signaling pathways of calcium, but to know whether this elevation of intracellular calcium observed in cells
stimulated with VE was related to intracellular or extracellular stores. Therefore, Figs. 3 and 4 show that the depletion of
intracellular stores by blocking Ca2+ ATPase pumps through the action of TG, is diminished in cells that have been exposed to
VE in comparison to control cells, while the final Ca2+ concentration is equivalent. The explanation for this observed result is
that stores of intracellular calcium had been stimulated by VE and hence the resulting concentration increase is the result of
stored calcium drainage and not of extracellular input. These results allow us to infer that BT has an effect on intracellular
calcium but does not allow us to clarify how calcium has been stimulated. Further studies are needed with emphasis on Tris
Inositol Phosphate (IP3) and Ryanodine Receptor (RyR), as these are important biological triggers for intracellular calcium
pathways [17].
Calcium is an important second messenger and is involved in many cellular signaling processes; therefore the study of
intracellular calcium release is an important indicator for cellular response. The involvement of intracellular calcium
mobilization with implementation of BT may be a good start for experimental strategies and to provide an explanation for
previously reported results [11, 12].
V. CONCLUSIONS
These studies show that the application of pranic healing can have an effect on intracellular calcium and help understand
the effect of complementary therapies on cell regeneration. The article also proves that VE is not the result of motivation
induced in the patient, by avoiding the Placebo Controversy. It is important to emphasize the importance of this finding since
keratinocytes are part of 90% of the epidermis. The effect of pranic healing on intracellular calcium release, as a signaling
mechanism for cell regeneration, suggests that the results observed by other researchers [11], [12] associated with cell
regeneration may be mediated by this channel.
Pranic healing provides VE as a function of time and the effect of this energy produces a linear increase of intracellular
calcium up to 400ms after initial stimulation. This VE increases the amount of intracellular calcium, hence increasing the rate
of metabolism and acting as a powerful biocatalyst that can accelerate the regeneration rate. This idea is consistent with the
two laws of PH: the law of auto recovery and the law of vital energy, which are both combined to say that the physical body is
able to heal itself and uses vital energy for this purpose [3]. Beverly Rubik suggests that Biofields are extremely weak
electromagnetic fields capable of transmitting electromagnetic bioinformation [10]. The use of HaCaT Cells as a biosensor for
BT, should serve to test Rubiks or any other hypothesis about the nature of VE. Additional parameters of the VE effect should
be explored, e.g. is it time-dependent? distance-dependent? reproducible by other healers? In summary, the correlation,
relationship between BT and Ca2+ release needs to be strengthened.
ACKNOWLEDGMENTS
This work is partially supported with a grant from Venezuela Ministry of Science and Technology, called “Studies for the
scientific validation, the use of Pranic Healing as monotherapy or complementary therapy on conventional animal models.
Project PG-2007001522, Subproject 3. Dr. Ricardo Silva is member of the Prometeo Program, sponsored by the Secretariat for
Higher Education, Science, Technology and Innovation (Senescyt) in the Republic of Ecuador.
CONFLICT OF INTEREST
When this research was performed, Dr. Herrera was Medical - Scientific Advisor in the area of Urology and Central Nervous
System for GSK-Venezuela, however GSK was in no way involved or in any way related with the present research. Senescyt did
not provide any additional support for the development of this research. Experiments were performed independently and the
authors of the article in no way altered or modified the measured results.
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Ricardo Silva Caracas, July 20, 1971. Electronic Engineer, Universidad Simon Bolivar, Caracas, Venezuela, 1996.
M.Sc. Biomedical Engineering, Universidad Simon Bolivar, Caracas, Venezuela, 1998. Ph.D. Integrative Biosciencies,
Neuroscience Option + Ph.D. Minor in Bioengineering, The Pennsylvania State University, State College,
Pennsylvania, U.S.A., 2005.
He is member of the Prometeo Program from Secretariat for Higher Education, Science, Technology and
Innovation (Senescyt) in the Republic of Ecuador. He works at Senescyt Zone 5/8 and at University of Guayaquil
(UG). He is leading the Medical Informatics Research Group (PROMEINFO) at UG. He is former director of the
Integrative Biosciences “Prof. Luis Lara-Estrella” Laboratory at Universidad Simon Bolivar in Venezuela where this
research was performed. Dr. Silva is IEEE-Senior member, Certified Clinical Engineer, member of various
international organizations and committees.
Alvaro Herrera Caracas, October 11, 1980. He got degree in Biology, Major in Cell Biology. Faculty of Sciences.
Universidad Central de Venezuela/Venezuelan Institute of Scientific Research (IVIC), Venezuela, 2004. Master
Studies in Physiological Sciences. Luis Razetti School of Medicine, Physiology Department. Universidad Central de
Venezuela, 2006. Ph.D in Biological Sciences (HONOR MENTION). University of California, Los Angeles (UCLA)
U.S.A., /Universidad Simón Bolívar (USB) (Venezuela), 2011.
He was head at the Biophysics and Electrophysiology Laboratory, Universidad Simon Bolívar (USB), Venezuela.
Possess extensive experience as a Scientific Advisor in the pharmaceutical industry carrying out several projects,
seminars and Workshops with high impact within of LATAM. His research focuses in the excitation-contraction (EC)
coupling process in mammalian skeletal muscle using a combination of electrophysiological, molecular genetics, and
optical tools. The overall goal of this approach is to obtain an in-depth understanding the key role of the Chloride Channel in T Tubules of
skeletal muscle as well as the localization and physiological implications for congenital myotonia. Key articles have been published
regarding this topic.
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Hordep Velasquez Caracas, January 23, 1998. He got Superior Technician degree in rehabilitation, mention
occupational therapy, from “May Hamilton” Rehabilitation College at “Dr. Miguel Perez Carreño”, National
Rehabilitation Center, Caracas Venezuela. Complementary and alternative medicine (CAM) practitioner since 1993 in
different therapeutic modalities, including biofield therapies.
He is a counselor in biofield therapies for the Venezuela Ministry of Science and Technology, participating in the
project called: Studies for the scientific validation, of use of Pranic Healing as monotherapy or complementary
therapy on conventional animal models.
ResearchGate has not been able to resolve any citations for this publication.
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In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.
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Altered growth and differentiation and a highly abnormal karyotype are generally believed to be indicators for tumorigenic conversion of human cells. Inactivation of TP53 is supposedly one possible mechanism for accelerated genetic aberrations via reduced control of the genetic integrity. To examine the significance of this functional relationship, we investigated the long-term development of the spontaneously immortalized human skin keratinocyte line HaCaT, carrying UV-specific mutations in both alleles of the TP53 tumor suppressor gene. During >300 passages, proliferation, clonogenicity, and serum-independent growth potential increased. In addition, HaCaT cells gained anchorage independence and at late passages showed reduced differentiation. Karyotypic analysis up to passage 225 revealed a high frequency of translocations and deletions, with a particular increase during passages 30 and 50. Nevertheless, the HaCaT cells remained nontumorigenic when injected subcutaneously, and noninvasive in surface transplants in nude mice. By comparative genomic hybridization, we confirmed the karyotypically identified phase of increased chromosomal aberrations between passages 30 and 50. However, before and thereafter, the CGH profiles of the individual chromosomes were largely unchanged, demonstrating that those translocations—also maintained in later passages—did not cause a gross chromosomal imbalance. Thus, our data suggest that multiple changes often correlated with a “transformed phenotype,” including extensive karyotypic alterations and mutational inactivation of TP53, are well compatible with a nontumorigenic phenotype of the HaCaT cells, and that preserved chromosomal balance may be crucial for this stability during long-term propagation. Genes Chromosom. Cancer 19:201–214, 1997. © 1997 Wiley-Liss, Inc.
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Extracellular calcium concentrations markedly affect the pattern of proliferation and differentiation in cultured keratinocytes. When medium contains 0.1 mM calcium or above, the cells lose their proliferative ability, rapidly stratify, and terminally differentiate. Because 1,25(OH)2D3 (a modulator of Ca++ homeostasis) enhances the differentiation of keratinocytes, we investigated whether a link exists between 1,25(OH)2D3-induced release of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) from PtdIns 4,5-P2 and intracellular calcium [Ca++]i release from keratinocytes. Specifically, primary culture of keratinocytes were loaded with fluorescence dye Fura-2AM (10 microM) and changes in fluorescence intensity were monitored at the excitation wavelengths of 340 and 380 nm and emission wavelength of 505 nm. Additions of two agonists, 1,25(OH)2D3 (1.2 x 10(-9) M) and 13-Cis retinoic acid (0.2 x 10(-9) M), to dye-loaded keratinocytes induced rapid release of [Ca++]i, respectively, followed by gradual return to the prestimulated state. Addition of Ins(1,4,5)P3 (10 microM) to saponin-treated (leaky) keratinocytes also resulted in a rapid release of [Ca++]i. In contrast, the addition of inositol-1,3,4,5-tetrakisphosphate Ins(1,3,4,5)P4 at similar concentrations exerted negligible effect. Taken together, these results support the view that 1,25(OH)2D3-induced [Ca++]i release in keratinocytes may be via the Ins(1,4,5)P3-induced early release of intracellular [Ca++]i. This may explain, at least in part, 1,25(OH)2D3-enhanced keratinocyte differentiation.