Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage
Aim: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) — mediated photodynamic damage. Methods: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lymp...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2008
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| Назва видання: | Experimental Oncology |
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irk-123456789-1392312018-06-20T03:11:19Z Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage Gamaleia, N.F. Shishko, E.D. Gluzman, D.F. Sklyarenko, L.M. Uncategorized Aim: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) — mediated photodynamic damage. Methods: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji, Namalwa) and T-cell (MT-4, HUT-78) lines were inestigated. Diagnoses of chronic lymphoproliferative disorders were made on the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and immunocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The number of dead cells was determined in 20 h with trypan blue dye exclusion test. Results: The striking difference in responsiveness to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL — 62.06 ± 4.03%) were destroyed under the lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm2. However, it was virtually impossible to attain any appreciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm2). High sensitivity to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. Conclusions: 1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes as a possible simple and low-traumatic test for B-CLL screening among the elderly people. Цель: изучить чувствительность к фотодинамическому воздействию, опосредованному 5-аминолевулиновой кислотой (АЛК), нормальных и малигнизированных лимфоцитов человека. Методы: объектом исследования служили лимфоциты, выделенные с помощью градиента плотности фиколл-верографина из крови здоровых доноров (6) и гематологических больных (20) с различными формами лимфолейкозов, а также трансформированные лимфоциты В-клеточных (Raji, Namalwa) и Т-клеточных (МТ-4, HUT-78) линий человека. Диагноз хронического лимфопролиферативного заболевания ставили на основании морфологического, цитохимического и иммуноцитохимического исследования периферической крови и клеток костного мозга; иммунофенотипирование проводили с использованием панели моноклональных антител к дифференцировочным антигенам Т-, В-лимфоцитов, NK-клеток и АВС-АР-метода. Трансформированные лимфоциты клеточных линий культивировали в стандартной RPMI-1640 среде с 10% эмбриональной телячьей сывороткой. Для фотодинамического воздействия клетки инкубировали с 1,0–5,0 мМ АЛК в течение 4 ч и облучали гелий-неоновым лазером (длина волны – 633 нм), варьируя дозу от 10 до 150 Дж/см2 . Количество погибших клеток после дополнительной 20-часовой инкубации определяли с помощью теста с трипановым синим. Результаты: показано, что нормальные и малигнизированные лимфоциты человека резко отличаются по чувствительности к АЛК-опосредованному фотодинамическому действию (АЛК-ФД). Значительная часть лимфоцитов крови больных лейкозом (в среднем 62,06 ± 4,03% у 10 пациентов с В-клеточным хроническим лимфолейкозом, В-ХЛЛ) погибала при минимальных параметрах воздействия (1 мМ АЛК, доза облучения 25 Дж/см2 ), тогда как даже наивысшая доза (5 мМ АЛК, 150 Дж/см2 ) не влияла на жизнеспособность нормальных лимфоцитов. В опытах in vitro в Т-клеточных линиях отмечали больший процент гибели клеток, чем в В-клеточных. При этом линия Raji отличается парадоксальным отсутствием зависимости количества погибших клеток от дозы воздействия, что, по-видимому, связано с известным для этой линии дефектом сигнальных путей апоптоза. Обсуждаются вероятные механизмы установленных отличий между нормальными и малигнизированными лимфоцитами в контексте альтерации процессов синтеза клетками гема при малигнизации. Выводы: 1) впервые показано, что малигнизированные лимфоциты крови больных лимфолейкозом проявляют высокую чувствительность к АЛК-ФД при фактическом отсутствии такой чувствительности у лимфоцитов здоровых доноров; 2) озлокачествленные лимфоциты Т-клеточных линий более чувствительны к АЛК-ФД по сравнению с В-клеточными; 3) для клеток линии Raji установлена аномальная зависимость доза/эффект; 4) на основе полученных результатов предложен простой и малотравматичный тест для скрининга пожилых людей на наличие В-ХЛЛ. 2008 Article Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage / N.F. Gamaleia, E.D. Shishko, D.F. Gluzman, L.M. Sklyarenko // Experimental Oncology. — 2008. — Т. 30, № 1. — С. 65-69. — Бібліогр.: 27 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139231 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Uncategorized Uncategorized Gamaleia, N.F. Shishko, E.D. Gluzman, D.F. Sklyarenko, L.M. Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage Experimental Oncology |
| description |
Aim: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) — mediated photodynamic damage. Methods: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6) and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji, Namalwa) and T-cell (MT-4, HUT-78) lines were inestigated. Diagnoses of chronic lymphoproliferative disorders were made on the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and immunocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The number of dead cells was determined in 20 h with trypan blue dye exclusion test. Results: The striking difference in responsiveness to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL — 62.06 ± 4.03%) were destroyed under the lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm2. However, it was virtually impossible to attain any appreciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm2). High sensitivity to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. Conclusions: 1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes as a possible simple and low-traumatic test for B-CLL screening among the elderly people. |
| format |
Article |
| author |
Gamaleia, N.F. Shishko, E.D. Gluzman, D.F. Sklyarenko, L.M. |
| author_facet |
Gamaleia, N.F. Shishko, E.D. Gluzman, D.F. Sklyarenko, L.M. |
| author_sort |
Gamaleia, N.F. |
| title |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| title_short |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| title_full |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| title_fullStr |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| title_full_unstemmed |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| title_sort |
sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2008 |
| topic_facet |
Uncategorized |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/139231 |
| citation_txt |
Sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid-mediated photodynamic damage / N.F. Gamaleia, E.D. Shishko, D.F. Gluzman, L.M. Sklyarenko // Experimental Oncology. — 2008. — Т. 30, № 1. — С. 65-69. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT gamaleianf sensitivityofnormalandmalignanthumanlymphocytesto5aminolevulinicacidmediatedphotodynamicdamage AT shishkoed sensitivityofnormalandmalignanthumanlymphocytesto5aminolevulinicacidmediatedphotodynamicdamage AT gluzmandf sensitivityofnormalandmalignanthumanlymphocytesto5aminolevulinicacidmediatedphotodynamicdamage AT sklyarenkolm sensitivityofnormalandmalignanthumanlymphocytesto5aminolevulinicacidmediatedphotodynamicdamage |
| first_indexed |
2025-07-10T07:51:00Z |
| last_indexed |
2025-07-10T07:51:00Z |
| _version_ |
1837245524171816960 |
| fulltext |
Experimental Oncology ��� ������ ���� ��arc�� ����� ������ ���� ��arc�� ���arc�� ��� �� ��
Among t�e p�otosensitizing substances� utilized for
p�otodynamic t�erapy of tumors �PDT�� �-aminolevu-
linic acid �ALA� is t�e only one t�at is not a p�otosen-
sitizer proper but a metabolic precursor of an active
product protoporp�yrin IX �Pp IX� in w�ic� it converts
upon introduction into organism [1� �]. T�erefore� levels
of t�e Pp IX accumulation in different tissues may vary
depending on tissue metabolism. It was s�own t�at
in tumors t�e Pp IX formation from exogenic ALA was
more intensive t�an in normal tissues [�� 4] presumably
because malignant cells �ave �ig�er activity of rate-lim-
iting enzyme porp�obilinogen deaminase [�] and lower
activity of anot�er enzyme ferroc�elatase [�] controlling
under normal conditions t�e Pp IX overproduction by its
transformation to �eme [7].
Taking into account t�at p�otodynamic elimination
of malignant lymp�ocytes may potentially �ave some
important practical applications �e. g.� graft purging
from residual leukemic cells in t�e course of autolo-
gous bone marrow transplantation; PDT of cutaneous
T-cell lymp�omas�� we set t�e task of studying antileu-
kemic efficiency of ALA-mediated PDT using as model
targets bot� �uman leukemia cell lines and neoplastic
lymp�ocytes isolated from patients wit� different
forms of leukemia. T�e results obtained demonstrate
pronounced difference in resistance to ALA-PDT of
lymp�ocytes from leukemic patients compared to t�e
cells from normal subjects suggesting t�e possibility
to employ t�is difference as an additional marker of
lymp�oid malignancy.
MATERIALS AND METHODS
�ononuclear cells� �ereafter referred to as lymp�o-
cytes� were isolated from fres�ly drawn �eparinized
perip�eral blood of �� �aematologic patients� w�ic�
were treated in different Kiev clinics� and � �ealt�y
donors �� men and � women� median age �1 years�
range ���4�� by a density-gradient tec�nique �Ficoll-
sodium metrizoate d = 1.�77 g/cm� � [�]. Preliminary
clinical dia gnoses of c�ronic lymp�oproliferative
disorders were verified by aut�ors on t�e grounds of
morp�ological� cytoc�emical and immunocytoc�emi-
cal investigations of perip�eral blood and bone marrow
cells [�]. Immunop�enotype determination was carried
out by means of a panel of monoclonal antibodies
�DakoCytomation� Denmark� to differentiation antigens
of T lymp�ocytes �CD�� CD�� CD4� CD�� CD7� CD���
B lymp�ocytes �CD1�� CD��� CD��� CD4��� and NK
cells �CD1�� CD�7�� and by immunocytoc�emical ABC-
AP met�od [�]. From eac� patient an informed consent
to participate in t�e investigation was obtained.
SENSITIVITY OF NORMAL AND MALIGNANT HUMAN LYMPHOCYTES
TO 5-AMINOLEVULINIC ACID-MEDIATED PHOTODYNAMIC DAMAGE
N.F. Gamaleia*, E.D. Shishko, D.F. Gluzman, L.M. Sklyarenko
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology NAS of Ukraine, Kiev, Ukraine
Aim: To compare the sensitivity of normal and malignant human lymphocytes to 5-aminolevulinic acid (ALA) — mediated pho-
todynamic damage. Methods: Blood lymphocytes isolated by Ficoll-sodium metrizoate density gradient from healthy donors (6)
and hematologic patients (20) with different forms of lympholeukemia, and also transformed lymphocytes of human B-cell (Raji,
Namalwa) and T-cell (MT-4, HUT-78) lines were inestigated. Diagnoses of chronic lymphoproliferative disorders were made on
the grounds of morphological, cytochemical and immunocytochemical studies of peripheral blood and bone marrow cells, with
immunophenotype determination by monoclonal antibodies to differentiation antigens of T, B lymphocytes and NK cells and im-
munocytochemical ABC-AP method. Cells of leukemic B- and T-cell lines were cultured in standard RPMI-1640 medium. For
photodynamic treatment, the cells were incubated with ALA and then irradiated by a helium-neon laser (wavelength of 633 nm). The
number of dead cells was determined in 20 h with trypan blue dye exclusion test. Results: The striking difference in responsiveness
to ALA-mediated photodynamic treatment (ALA-PDT) between normal lymphocytes and cells isolated from lymphatic leukemia
patients was established. A bulk of leukemic cells (mean for 10 patients with B-CLL — 62.06 ± 4.03%) were destroyed under the
lowest ALA-PDT doses tested: 1 mM ALA, irradiation dose of 25 J/cm2. However, it was virtually impossible to attain any appre-
ciable damage of lymphocytes from healthy donors even with the highest treatment doses (5 mM ALA, 150 J/cm2). High sensitivity
to ALA-PDT of malignant lymphocytes was confirmed in experiments with human T- and B-cell leukemic cell lines, and in these
experiments, an anomalous reaction to the treatment of Raji cells was also detected. The mechanisms of the difference between
normal and malignant lymphocytes are discussed in terms of altered heme-synthesis processes in malignant cells. Conclusions:
1) It is shown for the first time that blood lymphocytes from lymphatic leukemia patients are highly sensitive to the damage with
ALA-PDT while lymphocytes of normal donors are practically not damaged. 2) Transformed lymphocytes of human T-cell lines
are more sensitive than lymphocytes of B-cell lines. 3) Lymphocytes of the Raji line display anomalous dose-effect dependence with
ALA-PDT. 4) It is proposed to evaluate the drastic difference in ALA-PDT responsiveness of normal and malignant lymphocytes
as a possible simple and low-traumatic test for B-CLL screening among the elderly people.
Key Words: blood lymphocytes, donors, leukemic patients, T- and B-cell lines, photodynamic treatment, 5-aminolevulinic acid,
B-CLL screening.
Received: December 12, 2007.
*Correspondence: Fax: 038 (044) 258-16-56
Abbreviations used: ALA — 5-aminolevulinic acid; B-CLL — B-cell
chronic lymphocytic leukemia; PDT – photodynamic therapy;
Pp IX – protoporphyrin IX; T-LGLL — large granular lymphocytic
leukemia, T-cell variant.
Exp Oncol ����
��� 1� �����
�� Experimental Oncology ��� ������ ���� ��arc��
B-cell lines Raji and Namalwa ��uman Burkitt lym-
p�oma� [1�� 11] and T-cell lines �T-4 ��uman T-cell
leukemia� [1�] and HUT-7� ��uman cutaneous T-cell
lymp�oma� [1�] were obtained from culture bank of
R.E. Kavetsky Institute for Experimental Pat�ology� On-
cology and Radiobiology and were cultured in standard
RP�I-1�4� medium supplied wit� 1�% fetal bovine
serum. For p�otodynamic treatment� cell suspen-
sions in Hanks’ balanced salt solution �pH = 7.�� were
prepared from cultures of leukemic cell lines in log
p�ase of growt� or lymp�ocytes fres�ly isolated from
�eparini zed blood. Solutions of ALA �Synbias� Ukraine�
were prepared immediately before experiments in
t�e buffe red salt solution� pH = 7.�. A cell suspension
�� · 1��/ml� was incubated for 4 � wit� 1.���.� m� ALA
under �7 °C and t�en irradiated by �elium-neon laser
�wavelengt� of ��� nm�. Irradiation doses varied from
1� J/cm� to 1�� J/cm� depending on cell resistance.
After t�e lig�t treatment� cells were incubated ��7 °C�
in RP�I-1�4� medium wit� fetal serum for additional
�� � for p�otodynamic alterations to fully develop. T�e
number of dead cells was determined by trypan blue
dye exclusion test.
RESULTS
Lymp�ocytes isolated from �ealt�y donors turned
out quite resistant to t�e p�otodynamic treatment wit�
ALA. As it is s�own in Table 1� application of t�e entire
range of tested doses up to 1�� J/cm�� t�at was � times
more t�an t�e dose served usually as an effective
standard in experiments wit� malignant lymp�ocytes
��� J/cm��� could not induce any significant deat�
of treated cells. Equally unsuccessful were attempts
to boost p�otodynamic alteration of t�e cells raising
concentration of ALA from 1 m� �used for evaluation of
lymp�ocytes killing in studies wit� leukemia patients�
to ��� m�.
On t�e contrary� lymp�ocytes obtained from pa-
tients wit� lymp�atic leukemia� yielded in general easily
to p�otodynamic damage �Table ��. In � from 1� suc�
patients �№ 1�7� 1�� 1��� more t�an ��% of cells died
under c�osen standard regimen of PDT treatment
�1 m� ALA� �� J/cm��. �ost �7� of t�ese � leukemia
cases were diagnosed by cell cytomorp�olo gy and
immunop�enotype as B-cell c�ronic lymp�ocytic
leukemia �B-CLL�. Among ot�er 7 cases� in � patients
wit� B-CLL �№ �� �� 1�� t�e number of dead cells
also approac�ed ��% �4�� 4�� 41%�. And only in re-
mainder 4 patients �№ 11 — �airy cell leukemia and
№ 14�1� — T-cell large granular lymp�ocyte leuke-
mia� t�e number of dead cells was low: �4.4; �.7; 4%
and 1�%� respectively.
For t�e sake of comparison� in t�e Table � t�ree cases
�№ 1����� are also given in w�ic� a preliminary clinical
diagnosis of leukemia was not confirmed by laboratory
investigations� and t�e cell response to p�otodynamic
treatment proved to be accordingly insignificant: �.7;
�.�% and 17.�% of dead cells� respectively.
In parallels wit� lymp�ocytes isolated from blood of
leukemia patients� resistance to ALA-PDT of leukemic
B-cell lines �Raji and Namalwa� and T-cell lines ��T-4
and HUT-7�� was studied . P�otodynamic vulnerability
of t�ese cultured neoplastic lymp�ocytes was also
found to be by far exceeding suc� of normal blood
lymp�ocytes �see Table ��. And as it is seen from t�e
Table �� among 4 cultures tested� Raji stood out for
its comparatively �ig� resistance to ALA-PDT w�ile
HUT-7� seemed to be t�e most responsive.
Table 1. ALA-PDT action on lymphocytes of healthy donors
Donors ALA concentra-
tion (mM)
Laser radiation
doses (J/cm2)
Number of dead
cells (%)
№ 1 1.0 10.0 1.0
1.0 18.0 1.0
1.0 25.0 1.0
№ 2 2.0 10.0 0.0
2.0 18.0 0.2
2.0 25.0 0.5
№ 3 3.0 10.0 1.0
3.0 18.0 1.0
3.0 25.0 1.0
№ 4 4.0 10.0 0.0
4.0 18.0 0.0
4.0 25.0 0.1
№ 5 2.0 50.0 0.5
№ 6 5.0 100.0 1.5
5.0 150.0 1.5
Control (untreated cells) 0.5
DISCUSSION
In addition to selective low-invasive destruction
of tumors� PDT may be instrumental in elimina-
tion of malignant lymp�oid cells� as it is illustrated
by bone marrow transplants purging from residual
leukemic cells wit� merocyanine �4� assisted PDT
[14]� a procedure proposed to improve efficiency of
leukemia patients’ t�erapy. In t�at case� selectivity of
malignant lymp�ocytes killing� as opposed to normal
lymp�ocytes and bone marrow progenitors� acquires
particular importance.
T�e data presented �ere s�ow t�at t�ere is a striking
difference in responsiveness to ALA mediated PDT of
blood lymp�ocytes obtained from �ealt�y subjects and
leukemia patients. W�ereas in most cases� a bulk of
leukemic cells were quite easily destroyed by ALA-PDT
wit�out necessity to resort to �ig�er p�otosensitizer
concentrations or lig�t radiation doses �see Table ��
№ 1�1�� 1�� 1��� it was virtually impossible to attain any
appreciable damage of normal lymp�ocytes even wit�
t�e largest ALA-PDT doses tested. T�e contrasting re-
actions observed may be explained by peculiarities of
�eme metabolism in malignant cells. Heme synt�esis
from glycine and succinyl CoA under normal conditions
is a subject of t�e negative feedback regulation [7]:
an increase in t�e �eme formation in�ibits activity of
a rate limiting enzyme aminolevulinat- synt�ase and
decreases ALA concentration. If exogenic ALA is in-
troduced in organism t�e limiting link is passed round�
and t�e process proceeds down t�e c�ain to synt�esis
of Pp IX. In normal lymp�ocytes� t�e Pp IX by means of
ferroc�elatase joins Fe�+ being transformed to �eme
t�at unlike PpIX �as no p�otosensitizing properties.
But in malignant cells� ferroc�elatase activity is low
[�]. W�at is more� in t�e cells� intracellular iron stores
are spent for DNA and cytoc�rome synt�esis [1�]. As
Experimental Oncology ��� ������ ���� ��arc�� �7��� ������ ���� ��arc�� �7�arc�� �7� �7 �7
a result� in malignant cells Pp IX is accumulated giving
rise to t�e cell p�otosensitivity.
It is of note t�at most of t�e cases analyzed �ere�
t�at s�owed considerable lymp�ocyte responsive-
ness to ALA-PDT� were constituted by B-cell leukemia
patients� including 1� patients wit� B-CLL. Ot�er four
cases were a patient wit� T-cell c�ronic lymp�atic leu-
kemia �see Table �� № 1�� and t�ree patients wit� Т-cell
large granular lymp�ocyte leukemia �see Table ��
№ 14�1��. Curiously enoug�� t�ese four cases occu-
pied extreme �but opposite� positions on t�e general
�for studied leukemia patients� scale of ALA-PDT lym-
p�ocyte responsiveness: t�e first one wit� t�e largest
portion of cells killed ���%� and t�e ot�er t�ree — wit�
t�e smallest ��.7; 4; 1�%�. It remains to wait until more
patients wit� t�ese leukemia types are analysed to de-
cide w�et�er suc� reactivity levels are typical of t�em.
As to B-CLL patients� it seems t�at more t�an 4�% kill-
ing of lymp�ocytes �mean for 1� patients is �����% ±
4���%� under standard conditions c�osen is quite
c�aracteristic. Given relatively �ig� incidence of B-CLL
among t�e elderly population [1�]� drastic distinctions
in lymp�ocytes ALA-PDT responsiveness between t�e
patients and �ealt�y people s�ould be evaluated as
a possible simple and low-traumatic test for B-CLL
screening� especially if it is establis�ed t�at t�ere is
no essential overlap in t�e cell reactivity wit� reactive
lymp�ocytosis �t�e work in progress�. In t�is connec-
tion� t�ree cases wit� negative diagnoses� deliberately
included in Table � �№ 1������ are demonstrative. As
it �appened� ALA-PDT responsiveness tests wit� t�e
patients were carried out before immunocytological
investigation of t�e material �responsibility of anot�er
laboratory� was accomplis�ed� and strong doubts were
cast on t�e preliminary leukemia diagnosis forestalling
a formal diagnostic conclusion.
It was interesting to find out w�et�er leukemic lym-
p�ocytes� cultured as establis�ed cell lines� retain t�eir
�ig�tened ALA-PDT responsiveness in comparison to
normal cells. T�e data obtained wit� two B-cell lines
�Namalwa and Raji� and two T-cell lines ��T-4 and
HUT-7�� witness t�at it is t�e case �Table �� and cor-
roborate t�e findings of ot�er aut�ors [17���]� t�oug�
all of t�em worked wit� some ot�er leukemic cell lines.
From Table � it also appears t�at among four cell lines
studied� t�e Raji is distinctly distinguis�ed by its rela-
tively low responsiveness� surprisingly independent of
ALA-PDT doses applied. Considering t�e leading role
apoptotic mec�anisms play in p�otodynamic damage
of tumors [�4]�t�e anomalous reaction of Raji cells is
presumably connected wit� altered apoptosis pat�-
ways discovered in t�e cells [��]. Taken as a w�ole�
two T-cell lines tested are more ALA-PDT responsive
t�an B cell lines �could t�at be correlated to extremely
�ig� cell responsiveness of t�e only T-cell leukemia
patient analysed �see Table �� № 1��?�.
To t�e best of our knowledge� t�ere are no publis�ed
data on t�e ALA-PDT responsiveness of lymp�ocytes
from patients wit� lymp�atic leukemia. In a paper [��]�
preferential ALA-PDT killing of malignant lymp�ocytes
from a patient wit� cutaneous T-cell lymp�oma com-
pared to normal perip�eral blood lymp�ocytes was
described. Hrkal`s group reported [�7] t�at in � out
of 1� patients wit� acute myeloid leukemia� ALA-PDT
treatment of mononuclear cell preparations resulted in
substantial reduction of blast cells number w�ereas t�e
viability of normal lymp�ocytes was little affected.
Table 3. ALA-PDT action on leukemia cell lines
Cell lines ALA concentra-
tion (mM)
Laser radiation
doses (J/cm2)
Number of dead
cells (%)
B-cell lines
Raji 1.0 25 18.25 ± 1.7
2.5 50 15.3 ± 2.3
4.0 100 17.0 ± 2.0
Namalwa 1.0 25 36.0 ± 5.7
2.5 50 63.5 ± 2.5
3.0 75 97.6 ± 3.1
T-cell lines
MT-4 1.0 25 54.8 ± 3.4
2.5 50 96.3 ± 4.8
HUT-78 1.0 10 74.3 ± 2.3
1.0 25 95.7 ± 0.8
Table 2. ALA-PDT action on lymphocytes of patients with hematological diseases
Pa-
tients Diagnoses Phenotype (positive cells, %) Number of
dead cells (%)CD19 CD20 CD23 CD43 CD2 CD3 CD4 CD5 CD7 CD8 CD16 CD57
1 B-cell chronic lymphatic leukemia (B-CLL) 80 85 65 7 7 55 7 7 73.5 ± 2.36
2 B-CLL 90 90 90 5 Single
cells
95 Single
cells
Single
cells
61.8 ± 5.48
3 B-CLL 80 80 70 12 7 90 7 7 53.0 ± 2.1
4 B-CLL/non-Hodgkin`s lymphoma 50 50 54 80 37 82 60.3 ± 7.74
5 B-CLL 90 90 80 5 Single
cells
90 Single
cells
Single
cells
85.4 ± 4.09
6 B-CLL 75 70 60 80 15 67.2 ± 4.42
7 B-CLL 85 75 65 20 50 72.8 ± 4.66
8 B-CLL 70 64 50 24 57 48.0 ± 2.03
9 B-CLL 70 75 26 24 13 52 12 12 45.0 ± 2.16
10 Non-Hodgkin`s lymphoma (early leuke-
mization phase)
65 70 30 18
15 15 53.6 ± 2.4
11 Hairy cell leukemia 50 53 -/+ 43 43 42 43 43 24.4 ± 2.68
12 B-CLL 55 60 50 37 20 55 18 17 41.0 ± 2.7
13 T-cell chronic lymphatic leukemia 95 90 88 80 0 90.0 ± 3.5
14 Large granular lymphocytic leukemia,
T-cell variant (T-LGLL)
99 90 90 68 36 3.7 ± 1.03
15 T-LGLL 85 90 90 90 25 10 8 4.0 ± 0.5
16 T-LGLL 93 95 10 25 77 63 79 16.0 ± 1.76
17 Langerhan`s cell histiocytosis 11 11 73 30
30 34
20.5 ± 1.84
18 Polyclonal B-lymphocyte proliferation 33 28 60 5.7 ± 1.2
19 Polyclonal B-lymphocyte proliferation 21 17 84 78 6.6 ± 0.24
20 Polyclonal B-lymphocyte proliferation 17.2 ± 1.96
�� Experimental Oncology ��� ������ ���� ��arc��
ACKNOWLEDGEMENTS
T�is study was partly supported by National Acade-
my of Sciences grant ��1��U������� Ukraine.
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Experimental Oncology ��� ������ ���� ��arc�� ����� ������ ���� ��arc�� ���arc�� ��� �� ��
ЧУВСТВИТЕЛЬНОСТЬ НОРМАЛЬНЫХ
И МАЛИГНИЗИРОВАННЫХ ЛИМФОЦИТОВ ЧЕЛОВЕКА
К ФОТОДИНАМИЧЕСКОМУ ВОЗДЕЙСТВИЮ,
ОПОСРЕДОВАННОМУ 5-АМИНОЛЕВУЛИНОВОЙ КИСЛОТОЙ
Цель: изучить чувствительность к фотодинамическому воздействию, опосредованному 5-аминолевулиновой кислотой (АЛК),
нормальных и малигнизированных лимфоцитов человека. Методы: объектом исследования служили лимфоциты, выделенные
с помощью градиента плотности фиколл-верографина из крови здоровых доноров (6) и гематологических больных (20)
с различными формами лимфолейкозов, а также трансформированные лимфоциты В-клеточных (Raji, Namalwa) и Т-клеточных
(МТ-4, HUT-78) линий человека. Диагноз хронического лимфопролиферативного заболевания ставили на основании
морфологического, цитохимического и иммуноцитохимического исследования периферической крови и клеток костного мозга;
иммунофенотипирование проводили с использованием панели моноклональных антител к дифференцировочным антигенам
Т-, В-лимфоцитов, NK-клеток и АВС-АР-метода. Трансформированные лимфоциты клеточных линий культивировали в
стандартной RPMI-1640 среде с 10% эмбриональной телячьей сывороткой. Для фотодинамического воздействия клетки
инкубировали с 1,0–5,0 мМ АЛК в течение 4 ч и облучали гелий-неоновым лазером (длина волны – 633 нм), варьируя дозу от
10 до 150 Дж/см2 . Количество погибших клеток после дополнительной 20-часовой инкубации определяли с помощью теста
с трипановым синим. Результаты: показано, что нормальные и малигнизированные лимфоциты человека резко отличаются по
чувствительности к АЛК-опосредованному фотодинамическому действию (АЛК-ФД). Значительная часть лимфоцитов крови
больных лейкозом (в среднем 62,06 ± 4,03% у 10 пациентов с В-клеточным хроническим лимфолейкозом, В-ХЛЛ) погибала
при минимальных параметрах воздействия (1 мМ АЛК, доза облучения 25 Дж/см2 ), тогда как даже наивысшая доза (5 мМ
АЛК, 150 Дж/см2) не влияла на жизнеспособность нормальных лимфоцитов. В опытах in vitro в Т-клеточных линиях отмечали
больший процент гибели клеток, чем в В-клеточных. При этом линия Raji отличается парадоксальным отсутствием зависимости
количества погибших клеток от дозы воздействия, что, по-видимому, связано с известным для этой линии дефектом сигнальных
путей апоптоза. Обсуждаются вероятные механизмы установленных отличий между нормальными и малигнизированными
лимфоцитами в контексте альтерации процессов синтеза клетками гема при малигнизации. Выводы: 1) впервые показано,
что малигнизированные лимфоциты крови больных лимфолейкозом проявляют высокую чувствительность к АЛК-ФД
при фактическом отсутствии такой чувствительности у лимфоцитов здоровых доноров; 2) озлокачествленные лимфоциты
Т-клеточных линий более чувствительны к АЛК-ФД по сравнению с В-клеточными; 3) для клеток линии Raji установлена
аномальная зависимость доза/эффект; 4) на основе полученных результатов предложен простой и малотравматичный тест
для скрининга пожилых людей на наличие В-ХЛЛ.
Ключевые слова: лимфоциты крови, здоровые доноры, больные лейкозом, клеточные линии, фотодинамическое воздействие,
5-аминолевулиновая кислота, скрининг В-ХЛЛ.
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