(2023) 24:33
Iriani et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00495-0

BMC Molecular andCell Biology

Open Access

RESEARCH

TNFα induces Caspase-3 activity
in hematopoietic progenitor cells CD34+,
CD33+, and CD41 + of myelodysplastic
syndromes
Anggraini Iriani1* , Andhika Rachman2 , Rahayuningsih D. Setiabudy3, Siti B. Kresno3, Aru W. Sudoyo2,
Mansyur Arief4, Alida R. Harahap3 and Marsya Kaila Fatina5

Abstract
Background Cytopenia is the primary feature of Myelodysplastic Syndrome, even in the presence of hypercellularbone marrow. TNFα is recognized as both a proinflammatory, and proapoptotic cytokine with a well establishedrole in promoting apoptosis in MDS. Therefore, TNFα has the potential to be a valuable biomarker for predicting
the progression of cytopenia in MDS. This study aims to establish the role of TNFα exposure in triggering apoptosis
through caspase-3 activity in CD34+, CD33+, and CD41 + cells in MDS.
Methods This study is an in vitro comparative experimental research. Bone marrow mononuclear cells were isolated as the source of hematopoietic progenitor cells. Subsequently, CD34+, CD33+, and CD41 + cells were exposed
to rhTNFα, and the caspase-3 activity was measured using flowcytometry.
Results In MDS CD33 + and CD41 + caspase-3 activity of rhTNFα exposed cells was significantly higher than without exposed cells. The opposite result was found in CD34 + cells, where the caspase-3 activity without rhTNFα
exposed cells was significantly higher than rhTNFα exposed cells.
Conclusion rhTNFα exposure led to an elevation in caspase-3 activity in MDS progenitor cells, especially in thosethat had differentiated into myeloid cell CD33 + and megakaryocyte cell CD41+, as opposed to the early progenitor
cells CD34+.
Keywords Myelodysplastic syndrome, TNFα, caspase-3, CD34+, CD33+, CD41+*Correspondence:
Anggraini Iriani
anggraini.iriani@yarsi.ac.id1
Department of Clinical Pathology, Faculty of Medicine, Yarsi University Yarsi Hospital, Jl. Letjen Suprapto Kav 13, Cempaka Putih, Jakarta 10510,
Indonesia
2
Division of Hematology and Medical Oncology, Department of Internal
Medicine, Faculty of Medicine, Universitas Indonesia - Dr. Cipto
Mangunkusumo General Hospital, Jakarta, Indonesia
3
Department of Clinical Pathology, University of Indonesia, Jakarta,
Indonesia
4
Department of Clinical Pathology, Hasanuddin University, Makasar,Indonesia
5
Faculty of Medicine, Andalas University, Padang, Indonesia© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

Introduction
Myelodysplastic syndrome (MDS) is a heterogeneous
clonal stem cell disorder characterized by abnormalities
in the differentiation and proliferation of hematopoietic
precursors which leads to peripheral blood cytopenia,
bone marrow hypercellularity, ineffective hematopoiesis,
and dysplastic changes in myeloid, erythroid, and megakaryocyte lineages [1–4]. Cytopenia is the prominent
feature of MDS amid the hypercellularity in the bonemarrow [5, 6]. Some experts postulate that ineffective
hematopoiesis in MDS may be caused by the increase inhematopoietic cell apoptosis, coupled with heightened
intra-medullary proliferation or self-renewal capacity [7].
The bone marrow’s microenvironment is a highly intricate structure that plays a crucial role in regulating theproliferation, differentiation, and self-renewal processesof progenitor cells by providing essential factors such as
nutrients, growth factors, and cytokines. However, in
MDS, the bone marrow microenvironment undergoeschanges that involve interactions between various cell
types and between cells and cytokines, which contributeto the pathogenesis of the MDS [8, 9].
There are several hypotheses explaining the heightened occurrence of apoptosis. Firstly, increased apoptosis serves as a mechanism through which hematopoiesis
cells dispose of undesired cells. When bone marrow progenitor cells come across chronic peripheral cytopenia,
the body’s feedback mechanism triggers the productionof cytokines to facilitate the formation of a new cell.
Secondly, the elevation in apoptosis can be attributed toclonal abnormalities in MDS, implying that apoptosis
is not solely an outcome but is intricately linked to theunderlying cause or mechanism of abnormal biology in
MDS clones [10, 11].
The TNFα gene is located on chromosome 6p21.3 andspans a length of 3 kb, encompassing four exons. The
final exon is responsible for encoding approximately 80%of the secreted TNFα. TNFα itself is a type 2 transmembrane protein, composed of 212 amino acids and forma homotrimer structure. Various cell types, including
macrophages, monocytes, T cells, B cells, NK cells, andKupffer cells, produce TNFα. Its production is induced in
response to cytokine signals, such as GMCSF, IL-1, IL-2,
IFNγ, and immune complexes [12].
Apoptotic activity is assumed to be the primary mechanism underlying ineffective hematopoiesis, leading tocytopenia in the peripheral blood of MDS patients [8,13]. In these patients, there is a known elevation in thelevel of TNFα, a cytokine that triggers apoptosis via theextrinsic pathway [14]. TNFα interacts with the TNF
receptor initiating the activation of the caspase cascade.
Caspase is an intracellular proteolytic enzyme responsible for triggering apoptosis [15]. Specifically, activation ofPage 2 of 9

caspase-3 triggers a cell to initiate apoptosis. The activityof this enzyme is used as a marker for apoptotic signaling in cells. However, the exact location within MDS progenitor cells where this TNFα-induced apoptotic process
occurs remains unknown, whether it takes place in theearly progenitor cells (CD34 +) or in the differentiatedmyeloid progenitor cells (CD33 +) and megakaryocyte
progenitor cells (CD41+) [10, 11].
This study aims to demonstrate the impact of TNFα
exposure on inducing apoptotic activity in hematopoietic progenitor cells, specifically CD34+, CD33+, andCD41 + cells in MDS. We aim to show that this exposure
leads to an increase in caspase-3 activity particularly incells that have undergone differentiation into myeloid,
and megakaryocyte lineages.

Materials and methods
This study employed a comparative in vitro experimental design involving the isolation of bone marrow mononuclear cells (BMMC) from 15 MDS bone marrowaspirates. Diagnosis of MDS was established based onclinical examination, complete hematological examination, peripheral blood and bone marrow morphology
analysis [15], as well as cytogenetic testing according
to the International System for Human Cytogenomic
Nomenclature (ISCN) recommendations to align withthe 2016 WHO diagnostic criteria [2]. The samples usedconsisted of CD34+, CD33+, and CD41 + cells that wereisolated from the BMMC obtained from MDS patients.
As a control group, we isolated BMMC from 8 lymphomabone marrow aspirates, because it was not feasible toobtain bone marrow aspirates from healthy individuals,
and lymphoma typically exhibits abnormalities in nonmyeloid lineages, Table 1.
BMMC isolation

Isolation of BMMC was performed using density gradient centrifugation with Ficoll-Paque medium (GEHealthcare™, Sweden). A cell density of 300,000 BMMC
in 300 µL of StemMACS HSC media (Miltenyi Biotec™,USA) was used in each exposure. Cells were preparedin separate tubes, with some exposed to 48ng/100mL
recombinant human TNFα / rhTNFα (R&D systems™,USA) followed by incubation at ­37OC with 5% ­CO2 for
18 h. The remaining cells received the same treatment
without exposure to TNFα,
Caspase 3 activity measurement

The BMMC from both the MDS and control groups
underwent identical treatments, involving exposure to
TNFα and no exposure. Each group was examined for
caspase-3 activity, both with and without exposure to
the TNFα. Then, the delta (Δ ) caspase-3 activity was

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

Page 3 of 9

Table 1 Characteristic of MDS bone marrow aspirate

process which is though to be mainly induced by TNFα
was tested by exposing rhTNFα to CD34+, CD33+, andCD41+, to find out in which progenitor lineage the apoptosis process occurs more dominantly by examining caspase-3 activity, Fig. 1.

Bone marrow
Aspirate

Bone Marrow
Morphology

IPSS-R(score)MDS 1

MDS-SLDHigh (5)MDS 2

MDS-SLDIntermediate (4,5)

MDS 3

MDS-SLD

Very high (7)MDS 4

MDS-SLDNA

MDS 5

MDS-MLDHigh (4,5)

MDS 6

MDS-MLD

Low (2,5)

MDS 7

MDS-MLDIntermediate (4,5)

MDS 8

MDS-MLD

Very low (1)MDS 9

MDS-MLDNA

MDS 10MDS-MLDIntermediate (3,5)

MDS 11MDS-MLDNA

MDS 12

MDS-EB-1

Very high (7,5)

MDS 13

MDS-EB-1

Intermediate (4)MDS 14

MDS-EB-1

Intermediate (4,5)

MDS 15

MDS-EB-1

High (6)MDS Myelodysplastic syndrome, MDS-SLD Single lineage dysplasia, MDSMLD Multilineage dysplasia, MDS-EB-1 Excess blast 1, NA Not applicable, IPSS-R
international prognostic scoring system revised

Table 2 Caspase-3 examination panel
Tubea

Treatment

Fluorochrome antibody

1

Without exposure

Negative control

2

Cisplatin 100 µg/(10 µL)

Positive control

3

Without exposure

CD34 APC, CD40 PE, CD154 FITC

4

Without exposure

CD34 APC, Caspase-3 FITC

5

rhTNFα 48 ng

CD34 APC, Caspase-3 FITC

6

Without exposure

CD33 PE, Caspase-3 FITC

7

rhTNFα 48 ng/well

CD34 APC, CD33 PE, Caspase-3 FITC

8

Without exposure

CD41 PE, Caspase-3 FITC

9

rhTNFα 48 ng/well

CD34 APC, CD41a PE, Caspase-3 FITC

10

Without exposure

CD73 PE, Caspase-3 FITC

11

rhTNFα 48 ng/well

CD34 APC, CD73 PE, Caspase-3 FITCa

Each tube contains BMMC with density of 300,000 cells/300µL media

measured, representing the difference between caspase-3
activity in BMMC exposed to rhTNFα and those without
exposure. The BMMC were then labeled with specific fluorochrome antibodies; (CD34 + with APC, CD33 + withPE, and CD41 + with PE); and a fluorochrome FITC label
was applied to assess caspase-3 activity (BD™, USA). The
fluorescence intensity was quantified using the BD FACSCalibur flowcytometry, Table 2.
The conceptual framework of this study is the BMMC
from MDS patients, consist of CD34+, CD33+, andCD41 + in microenvironment of bone marrow there areconditions that support proapoptotic. This study tries
to look at these in vivo condition in vitro. The apoptosis

Flowcytometry analysis

Measurement of active caspase-3 was conducted using
flow cytometry, which involves passing cells (events)
through a detector. A number of cells were examined andanalyzed on a scatter diagram. The x-axis represented theintensity of FITC against caspase-3 in the cells, while they-axis described the fluorescence intensity against cellmarkers (CD34, CD33, CD41). Cells displaying a fluorescent pattern were identified and compared with theentire cell population, after which the percentage wascalculated.
Statistical analysis

To analyze the data for differences between paired groups(those without exposure and those exposed to rhTNFα) apaired t-test was employed, whereas, for unpaired groups(comparing the MDS group to the control group) an
independent t-test was used.
The comparison of ∆ caspase-3 activity between the
CD34+, CD33+, and CD41 + cell groups in MDS andthe corresponding control cell groups was conducted.
Both groups under conditions without exposure andafter exposure to rhTNFα. A one-way ANOVA test was
performed for more than two unpaired groups. If theresults of the One-Way ANOVA test indicated a p-valueof < 0.001. Therefore, it can be concluded that there wereat least two distinct variations in the caspase-3 activity
mean among the progenitor cell groups, which were significantly different in both the MDS and control groups.

Results
The mean of Δ activity caspase-3 in CD34 + between
MDS and control groups showed that there was a significant difference with p-value 0.016. The paired t-test to
analyze caspase-3 activity in CD34 + MDS without exposure and those exposed to rhTNFα showed a significant
difference with p-value 0.034, as well as in control withp-value 0.008. In the MDS group the mean of caspase-3
activity in CD34 + without exposure (22.67%) was higher
than in CD34 + exposed to rhTNFα (19.53%), with a negative Δ activity caspase-3. In control group the mean caspase-3 activity in CD34 + exposed to rhTNFα (23.72%)
was higher than without exposure (18.84%), Fig. 2. Thisshows that CD34 + has weird behavior because even
without exposure to rhTNFα the caspase-3 activity tends
to be higher.

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

Page 4 of 9

Fig. 1 The idea is that exposure of rhTNFα to the BMMC of MDS, CD34+, CD33+, and CD41 + would induce apoptosis through extrinsic pathway
via TRADD and activate cascade caspase to increase caspase-3 activity

Fig. 2 The caspase-3 activity in MDS and control BMMC unexposed and exposed to rhTNFα. A Caspase activity in CD34 + cells, B Caspase-3
activity in CD33+, C Caspase activity in CD41 + cells. Data presented in mean (SD). Blue box: MDS group; green box: control group. Left side:
the group of cells unexposed to rhTNFα; middle side: the group of cells exposed to rhTNFα; right side: delta value. Delta formula: caspase-3 activity
after exposure rhTNFα subtracted by caspase-3 activity before exposure

The mean of Δ activity caspase-3 in CD33 + between
MDS and control groups showed that there was a significant difference with p-value 0.006. The paired t test to
analyze caspase activity in CD33 + without exposure andexposed to rhTNFα showed a significant difference inboth groups, MDS with p-value < 0.001 and control withp-value 0.031. The paired t-test in CD41 + MDS without
exposure and exposed to rhTNFα showed a significant
difference with p-value 0.028. One of the results of caspase-3 activity shows in Fig. 3.

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

Page 5 of 9

Fig. 3 Caspase-3 Activity on MDS BMMC by Flowcytometry Analysis. A result of caspase-3 activity is indicated by gating in the upper right quadrant(UR). A CD34 + cell without exposure, B CD34 + cell exposed to rh-TNFα, C CD33 + cell without exposure, D CD33 + cell exposed to rh-TNFα,E CD41 + cell without exposure, F CD41 + cell exposed to rh-TNFα

Due to the statistical significance in the one-way
ANOVA test among the different cell group variants,
we proceeded to conduct Tamhane’s post hoc test to

identify which groups exhibit differences. The disparity
in mean Δ caspase activity is negative (-), indicating that
CD34 + caspase activity is lower than caspase-3 activity

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

in its comparator progenitor cells, namely CD33 + andCD41 + cells. A significant difference in mean Δ caspase-3 activity was observed within the MDS group,
specifically between CD34 + and CD33 + cells, as well as
between CD34 + cells and CD41 + cells, with p-values of< 0.001 and 0.013, respectively.

Discussion
Exposure to rhTNFα induced apoptosis in MDS
hematopoetic progenitor cells

The purpose of rhTNFα exposure is to induce these proapoptotic cytokines and observe the apoptotic response,
specifically caspase-3 activity in CD34+, CD33+, andCD41 + MDS BMMC. Apoptosis may occur througheither the intrinsic or extrinsic pathway. The TNFα apoptosis mechanism model operates through the extrinsic
pathway.
The TNFα signaling pathway mechanism for initiating
the apoptotic process begins with the binding of TNFα
to TNFR-1 and TNFR-2 receptors. TNFR-1 mediates
the cell death program by initially binding directly to theadapter protein TNF receptor-associated death domain(TRADD), then indirectly connecting to the FASassociated protein with a death domain (FADD). Thissequential process ultimately leads to the activation of
the caspase cascade, culminating in the execution phasefacilitated by caspase-3 [16–18].
Exposure of rhTNFα to hematopoietic progenitor
cells is expected to result in the binding of rhTNFα to
its receptor, TNFR1. This binding is anticipated to initiate cell death signals through the death-inducing signaling complex (DISC) and activate TRADD. Subsequently,
caspase activation will be triggered, ultimately leading to
the activation of caspase-3, which serves as the executorof apoptosis in hematopoietic progenitor cells in MDS, as
shown in Fig. 1.
The role of TNFα in triggering apoptosis is supported
by a research report that discovered a positive correlation between elevated serum TNFα levels and anemia in
MDS patients. Furthermore, an increase in TNFα levelsin the bone marrow was linked to heightened apoptosis during the early stages of MDS [19]. Several studies
have also documented increased activity of caspase-1 andcaspase-3 in hematopoiesis cells of MDS patients, with
caspase-3 activity being ten times higher than that of caspase-1. Notably, the heightened caspase-3 activity is positively associated with increased TNFα levels [20, 21].
However, it is possible for caspase-3 activation to
occur through interactions aside from rhTNFα exposure,although such instances were negligible in this study. We
determined this by calculating the delta value (Δ) for Caspase-3 using the formula: the caspase-3 activity data after
rhTNFα exposure subtracted by data before exposure

Page 6 of 9

(as the baseline). The positive and statistically significant
delta values indicate that rhTNFa exposure can indeed
increase caspase-3 activity.
The involvement of TNFα as an apoptosis trigger in
MDS was confirmed by the elevated levels of TNFα in theserum of MDS patients. This increase in TNFα showed apositive correlation with clinical anemia, and an elevatedrate of apoptosis in the early stages of MDS [19].
In MDS, there is an increasein the activity of both caspase-1 and caspase-3, with caspase-3 activity being tentimes higher than caspase-1 [7, 13, 14]. This increased
caspase-3 activity is associated with a high level of TNFα[20, 21]. Iriani et al. [22] discovered that the high levels of
TNFα in MDS are initiated by an increase in the expression of TNFα mRNA, triggered by sCD40L stimulation.
A substantial portion of MDS cells undergo intramedullary apoptosis, which can be challenging to detect morphologically, as it predominantly occurs at an early stage.
Therefore, the assessment of apoptosis in MDS should
involve early markers of apoptosis, such as caspase-3.
Apoptosis in CD34+

In Fig. 2A, the Δ caspase-3 value in the MDS group
was negative, whereas it was positive, with a significant
p-value. The Δ value was used to gauge the extent of the
impact of rhTNFα exposure. This suggests that, in the
MDS group, apoptosis had occurred in CD34 + cells even
without exposure to rhTNFα, in contrast to the control
group. This implies that CD34 + MDS cells naturally tend
to undergo apoptosis.
Reports indicate that CD34 + MDS cells tend toundergo apoptosis, with their susceptibility to apoptosis
being most pronounced among CD34 + progenitor cells.
[11, 23] This susceptibility is attributed to defects in theprogenitor stem cells. Due to these clonal abnormalities, daughter cells would develop into defective cells thatare prone to apoptosis. Another study found that MDS
CD34 + cells displayed higher apoptotic activity thannormal cells, with the apoptosis rate in MDS CD34 + cells
being 2.08 times faster than their proliferation rate [24].
In MDS, despite the bone marrow continuing to produce
cells, the cell cycle is shorter. It is also known that over
two-third of MDS cells at various stages of differentiationin the bone marrow undergo apoptosis.
Apoptosis in CD33+

In Fig. 2B, the ∆ caspase-3 value in the MDS group was
higher than that in the control group, with a significant
p-value. This suggests that the exposure of rhTNFα led
to increased apoptosis in both groups, but the impact ofincreasing apoptosis in CD33 + MDS cell group was moresignificant.

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

Raza et al. [11] found that in MDS, exposure of TNFα
would result in increased apoptosis in both CD33 + andCD34 + cells. In MDS, despite the excess apoptosis in
the bone marrow, granulocyte in the peripheral circulation tend to be more resistant to apoptosis. This occurs
because sensitive granulocytes undergo apoptosis earlier
in the bone marrow, while apoptosis-resistant granulocytes continue to enter the circulation. Apoptosis in the
myeloid lineage is also affected by myeloid-derived suppressor cells (MDSCs). In MDS, there is an increase incirculating MDSCs. MDSCs induce apoptosis in hematopoietic progenitor cells and trigger the secretion of suppressive cytokines, IL10 and TGFβ, which suppress thedevelopment of immature myeloid cells [25].
Apoptosis in CD41+

In Fig. 2C, caspase-3 activity in CD41 + in the MDS
group exposed to rhTNFα was significantly higher compared to the group without exposure. This indicated that
TNFα exposure to MDS megakaryocyte-platelet lineage cells leads to increased apoptosis. Cevic et al. [26],also observed that TNFα increased caspase-3 activity inplatelets.
Apoptosis in megakaryocyte-platelets lineage of MDS
can occur through the extrinsic pathway involving TNFα,Page 7 of 9

but it predominantly takes place via the intrinsic pathway. This is supported by the finding of higher levels of
Bax and Bak proteins [27], as well as the possibility of
mitochondrial activation or the release of cytochrome Cfrom the cytosol [28].
Following the maximum release of platelets (1000–
3000 platelets), senescent megakaryocytes, characterized
by a thin nucleus and cytoplasm, immediately undergo
apoptosis and are phagocytosed by macrophages [29].
The apoptosis in MDS megakaryocytes occurs due toineffective platelet production caused by the premature
apoptosis of mature megakaryocytes in the intramedullary. Cytogenetic abnormalities in MDS clones primarily led to defects in megakaryocytes, manifestingin morphological abnormalities, and disturbances innucleus differentiation and maturation. Moreover, theseabnormalities are influenced by the MDS bone marrow
microenvironment.
To address the hypothesis regarding whether apoptosis in MDS progenitor cell occurs during the early stage
or at a differentiation stage, a post hoc analysis was performed to examine the apoptotic activity of caspase-3
in CD34 + cells, representing early progenitors, and in
CD33 + and CD41 + cells, representing differentiatedprogenitors. This study demonstrates that exposure to

Fig. 4 Scheme of Apoptosis Mechanism on MDS Hematopoesis Progenitor Cell by. TNFα. Early hematopoesis progenitor cell express
CD34 + surface marker. Then it will differentiate into specific myeloid progenitor (CD33+), megakaryocyte-thrombocyte progenitor (CD41+).
This image illustrates that TNFα induces apoptosis in MDS hematopoesis progenitor cell, especially to cell that have differentiated into myeloid
CD33 + and megakaryocyte-thrombocyte CD41+

Iriani et al. BMC Molecular and Cell Biology(2023) 24:33

rhTNFα significantly increases caspase-3 activity, especially in hematopoietic progenitor cells that differentiate
into the myeloid lineage CD33 + and the megakaryocytethrombocyte lineage CD41+, as opposed to early progenitor lineage CD34+, Fig. 4.
These findings shed light on why cytopenia in MDS ismore pronounced than the increase in blast cells. Similar results were reported by Xia et al. [30], who examined
apoptotic activity in MDS CD34+ (progenitor cells) andMDS CD34- (mature cells), and found higher apoptotic
activity in mature cells. However, their study did not
distinguish the specific stage of differentiation among
mature cells.
These results contrast with the previous hypothesis,
which suggested that apoptosis in MDS mainly occurs inearly progenitor cells CD34+.
The reason MDS develops slowly but progressively liesin the fact that CD34 + MDS cells are long-term repopulating stem cells (LT-HSC) with a high self-renewal
capacity but a slow proliferation rate. The MDS cell clone
could persist for up to 17 years without clinically significant changes in bone marrow or peripheral blood. However, after prolonged perios, the bone marrow exhibits
an 8.15% presence of dysplastic progenitor cells. Theseabnormal progenitor cells would significantly reduce thenumber of mature cells in the peripheral blood, leading
to cytopenia [31].
This explanation helps clarify the typical phenomenon
in MDS, where there is hypercellularity in the bone marrow but cytopenia in the peripheral blood. It suggeststhat apoptosis occurs mainly in differentiated progenitor
cells rather than in early progenitor cells. Unlike othermalignant cells that can further develop, MDS clonalcells fail to mature, leading to an accumulation of cellswith increased apoptotic abilities.

Page 8 of 9

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​023-​00495-0.
Additional file 1: Supplementary Table 1 . Characteristics of The Study
Subjects. Supplementary Table 2. Caspase-3 Activity in CD34+, CD33+
and CD41+ Cells Without exposure and exposure to rhTNFa. Supplementary Table 3. One Way Anova test and Post Hoc test to D Caspase-3 in
CD34+, CD33+ and CD41+ Cells.
Acknowledgements
Not applicable.
Authors’ contributions
AI, RDS, SBK AWS conceptualization and design the research; AI, AR, RDS,
ARH, MKF collected samples, data analysis and interpretation; AR, RDS, SBK,AWS, MA, MKF critical revision and review manuscript. The authors read andapproved the final manuscript.
Funding
Part of this research was founded by the Research Development of Medical Science and Technology, Ministry of Health Republic of Indonesia noLB.02.01/I.1/4676.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
in our institutionalized database and the Copy Right of this study has been
deposited in the repository of Yarsi University, or please contact the corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
This study has received ethical approval from the Ethics Committee of the Faculty of Medicine University of Indonesia No.673/UN2.F1/ETIK. This study was
conducted in accordance with the declaration of Helsinki. Moreover, informed
consent forms were duly signed by all participants.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 22 September 2022 Accepted: 8 November 2023

Conclusion
We have demonstrated that rhTNFα, a proapoptotic
cytokine, plays a significant role in initiating apoptotic
activity through caspase-3 in hematopoietic cells of MDS.
This effect is particularly pronounced in cells that havealready differentiated into myeloid lineage CD33 + andthe megakaryocyte-thrombocyte lineage CD41+, as
opposed to early progenitor cells CD34+. The results ofthis study have effectively substantiated the hypothesisthat apoptosis predominantly takes place in differentiatedprogenitor cells, specifically in the myeloid and platelet lineages, as opposed to early progenitor cells in MDS
hematopoiesis. This revised hypothesis differs from theinitial one, which suggested that apoptosis mainly occurs
in CD34 + early progenitor cells in MDS.

References
1. Tefferi A, Vardiman JW. Mechanisms of disease myelodysplastic syndromes. N Engl J Med. 2009;361:1872–85.
2. Arber DA, Orazi A, Hasserjian R, Thiele J, Borowitz MJ, Le Beau MM, et al.
The 2016 revision to the World Health Organization classification of
myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391–405.
3. Ma X. Epidemiology of myelodysplastic syndromes. Am J Med.
2012;125:S2.
4. Germing U, Neurkirchen J, Haas R. The epidemiology of myelodysplastic syndromes. Clin Leuk. 2008;2:34–8.
5. Valent P, Horny HP, Bennett JM, Fonatsch C, Germing U, Greenberg P,
et al. Definitions and standards in the diagnosis and treatment of the
myelodysplastic syndromes: Consensus statements and report from aworking conference. Leuk Res. 2007;31:727–36.
6. Bain BJ. Leukemia diagnosis. Wiley Blackwell; 2010.

Iriani et al. BMC Molecular and Cell Biology

7.

(2023) 24:33

Steensma DP, List AF. Genetic testing in the myelodysplastic syndromes: molecular insights into hematologic diversity. Mayo Clin Proc.
2005;80:681–98.
8. Aleshin A, Greenberg PL. Molecular pathophysiology of the
myelodysplastic syndromes insight for targeted therapy. Blood.
2018;2(20):2787–97.
9. Comont T, Treiner E, Vergez F. From immune dysregulations to therapeutic perspectives in myelodysplastic syndromes: a review. Diagnostic.
2021;11(11):1982.
10. Bennett JM, Kouides PA, Forman SJ. The myelodysplastic syndromes
morphology, risk assessment, and clinical management. Int J Hematol.
2002;76(Suppl 2):228–38.
11. Raza A, Gezer S, Mundle S, Gao X, Alvi S, Borok R, et al. Apoptosis inbone marrow biopsy samples involving stromal and hematopoietic cells in 50 patients with myelodysplastic syndromes. Blood.
1995;86(1):268–76.
12. Jang DI, Lee AH, Shin HY, Song HR, Park JH, Kang TB, et al. He role of TNF-α
in autoimmune disease and current TNF-α inhibitors in therapeutics. Int J
Mol Sci. 2021;22(5):2719.
13. D’Silva S, Rajadhyaksha SB, Singh M. Immune dysregulation in MDS: therole of cytokines and immune cells. In: Fuchs O, editor. Recent development in myelodysplastic syndromes. Intechopen. 2019. p. 1–17.
14. Sallman DA, List A. The central role of inflammatory signaling in the pathogenesis of myelodysplastic syndromes. Blood.
2019;133(10):1039–48.
15. Bain BJ, Bates I, Laffan MA, Lewis SM. Dacie and Lewis practical haematology. Elsevier Churcil Livingstone; 2012.
16. Gu C, Zhang J, Chen Y, Lei J. A trigger model of apoptosis induced bytumor necrosis factor signaling. BMC Syst Biol. 2011;5(Suppl 1):S13.
17. Webster JD, Vucic D. The balance of TNF mediated pathways regulates
inflammatory cell death signaling in healthy and diseased tissues. Front
Cell Dev Biol. 2020;8:365.
18. Loo GV, Bertrand MJM. Death by TNF a road to inflammation. Nat Rev
Immunol. 2023;23:289–303.
19. Kerbauy DB, Deeg HJ. Apoptosis and anti apoptotic mechanisms in the
progression of MDS. Exp Hematol. 2007;35:1739.
20. Ali A, Mundle SD, Ragasa D, Reza S, Shetty V, Mativi BY. Sequential activation of caspase 1 and caspase 3 like proteases during
apoptosis in myelodysplastic syndromes. J Hematol Stem Cell Res.
1999;8(4):343–56.
21. Mundle SD, Reza S, Ali A, Mativi BY, Shetty V, Venugopal P, et al. Correlation of tumor necrosis factor α (tnfα) with high caspase 3 like activity inmyelodysplastic syndromes. Cancer Lett. 1999;140(1–2):201–7.
22. Iriani A, Setiabudy RD, Kresno SB, Sudoyo AW, Bardosono S, Rachman A,
et al. Expression of mRNA TNFα and level of protein TNFα after exposure
sCD40L in bone marrow mononuclear cells of myelodysplastic syndromes. Stem Cell Investig. 2021;8(6):1–9.
23. Bachegonda L, Gligich O, Mantzaris I, Schinke C, Wyville D, Carrillo T,
et al. Signal transduction inhibitors in treatment of myelodysplastic
syndromes. J Hematol Oncol. 2013;6:1–11.
24. Parker JE, Mufti GJ, Rasool F, Mijovic A, Devereux S, Pagliuca A. The role ofapoptosis, proliferation, and the bcl-2–related proteins in the myelodysplastic syndromes and acute myeloid leukemia secondary to MDS. Blood.
2000;96:3932–8.
25. Fozza C, Crobu V, Isoni MA, Dore F. The immune landscape of myelodysplastic syndromes. Crit Rev Oncol Hematol. 2016;107:90–9.
26. Cevic O, Adiguzel Z, Baykal AT, Sener A. TNFα induced caspase 3 activation related inos gene expresion in adp activated platelet. Turk J Biol.
2017;41(1):31–41.
27. Josefsson EC, Vainchenker W, James C. Regulation of platelet production
and life span: role of Bcl-xL and potential implications for human platelet
diseases. Int J Mol Sci. 2020;165:1–12.
28. Kile BT. The role of apoptosis in megakaryocytes and platelet. Br J Haematol. 2014;165:217–26.
29. Radley JM, Haller CJ. Fate of senescent megakaryocyte in bone marrow.
Br J Haematol. 1983;53:227–87.
30. Xia B, Guo Q, Zhao D, Zhao HF, Han XP, Wang H, et al. Significance of
cd34- and cd34+ cell apoptosis and proliferation in bone marrow ofpatient with mds and the impact on survival. Zhongguo Shi Yan Xue Ye
Xue Za Zhi. 2012;20(6):1392–7.

Page 9 of 9

31. Walenda T, Stiehl T, Braun H, Frobel J, Ho AD, Schroeder T, et al. Feedback
signals in myelodysplastic syndromes increased self renewal of themalignant clone suppresses normal hematopoiesis. PloS Comput Biol.
2014;2014(4):1–9.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ready to submit your research ? Choose BMC and benefit from:• fast, convenient online submission• thorough peer review by experienced researchers in your field• rapid publication on acceptance• support for research data, including large and complex data types• gold Open Access which fosters wider collaboration and increased citations• maximum visibility for your research: over 100M website views per year
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions