WNT 

gene expression in B cells treated with retinoic acid

Ileana Santana and Jonathan Cordero, University of Puerto Rico, Department of Biology

Abstract

At the present time the retinoic acid has been long associate with immune system in
treatments of cancer with positives results. This has been demonstrated by
the proliferation of B lymphocytes an essential component of the adaptive immune system
in patients. In undertaking this work we deduce that the WNT genes would be active when
treated with retinoic acid. These genes work providing instructions and signaling that
promotes the growth, differentiation and proliferation of cells that fight antigens in body.
When analyzing the cellular density, with  spectrophotometric readings  and
images of microarrays, it was possible to obtain results with a tendency for the tumor
suppressor cells being up regulated by these genes. Genes like Wnt7b, Wnt1, Wnt8a and
Wnt8b de la familia WNT mostraba una tendencia a down regulated células proto-
oncogene mientras up regulated tumor supresor cells genes like Wnt2 and Wnt5a.
The WNT genes have the potential for study in the development and differentiation
of cancer cells treated with retinoic acid.

Background and significance

One type of white blood cells of great importance and scientific research, has been
the study of B lymphocytes. These cells play an important role in antibody production and
destruction of abnormal cells through specific receptors. This type of cell is produced in the
spinal cord and is in the blood and lymphatic tissue (Brandan, Fernanda, Luponio, &
Hope). Moreover, the discovery of vitamin A in its active form, retinoic acid, takes an
important part in researches that seek to counteract symptoms and diseases related to B
lymphocytes. Recently has been used to maximize the effectiveness of cancer
treatments. This is envident in the investigation of Wayne Szeto, Wen Jiang, David
A. Tice, et al, which have found that "Genetic Defects in the Wnt-1 signaling pathway
Contribute to human tumor progression and are especially prevalent in colorectal cancer”.
In that case they’ve screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and
localize a gene known to be up-regulated by retinoic acid. “The up-regulation of that gene
was also observed in hyperplastic mammary tissue and mammary gland tumors from
transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in
Wnt-1 signaling” ( W a y n e , W e n , D a v i d , B o n n e e , & F o n g , 2 0 0 1 ) . Also genes like tumor
necrosis factor family 4-1BB ligand, ephrin B1, Stra6, autotoxin and ISLR that encodes for
cell surface antigens were up-regulated by Wnt-1 and retinoic acid ( S o l o v i e v , F o n g , D a v i d ,
B o n n e e , & S m i t h , 2 0 0 2 ) . WNT family has about 19 genes that may be expressing cell
proliferation promoting proto-oncogene or tumor suppressor that can be up-
regulated or down Regulated by retinoic acid.

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Against this background it would be best to identify (in a random way)
which WNTfamily genes specifically expressed and what trends are presented in terms of
regulation of proto-oncogenes or tumor suppressor cells proliferation in the retinoic
acid treatment. Therefore, it is clear that by identifying these genes, the study would
be more focused on those genes with the characteristics we seek. Besides this,
the investigation of the activation and deactivation of these genes, could open the
door to more detailed investigations with more specific genes of WNT genes family, to
monitor the regulation of B cells under the treatment with retinoic acid.

Specific Aim

The purpose that drives us to present this work to determine the influence of retinoic

acid in the WNT gene expression in B lymphocyte cells. This would be determined by
the activation of the canonical pathway where protein axin, hijacked by an activate
membrane receptor (LRP) for a WNT gene family, avoid the degradation of β-
catenin protein, which operate in an adverse promoting cell proliferation proto-oncogene.
In comparisson to our DMSO treatment control, we expect to find in the B cell lymphocite
culture treated with retinoic acid a large number of expression in tumor supressor genes and
a decrease in expression in proto-oncogenes. WNT family genes may be responsible for
growth and development of the B cells or for inmune response, both parameters which are
influenced by retinoic acid intake.

Metodología

Bearing in mind the qualities of retinoic acid (RA) and Wnt gene expression, B
lymphocyte cells were treated with retinoic acid, using Iscove's Modified Dulbecco's
Medium (IMDM) in a flask of about 250ml and M12g3r cell suspension. It needed a
control group with the same conditions but without addition of RA, for comparison of
results. Were counted cell density cell culture via a hemacytometer. We Added Trypan
Blue in a small cell suspension using a sterile pipette to load the hemacytometer, which
taint dead cells that would not be counted to determine density. In order to meet the
immediate effect of RA in cell density, use was made of a sub culture with both solutions,
control and RA treatment. Incubated at 37 ° C in an atmosphere of 7.5% CO2 for two
days. After two days past, we used to determine the density of sub cell culture to compare
the difference in cell density. Then we extracted the total RNA of high quality, with the
help of a kit (RNeasy Plus Procedure) qualified for these processes. In first place must be
treated against nucleases DNA to prevent RNA degradation. The centrifugations after these
processes are very important because they help separate the different cellular components,
discarding those that do not need (supernatant). Through Buffer RLT Plus, it achieves one
of the most important steps in RNA extraction, disruption of plasma membranes. 

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Homogenization is another important step that helps to reduce the viscosity after disruption.
To discard the rest of DNA remaining in the samples, subjected to a purification process of
purification columns composed of silica (electrophoresis). The other solutions will
be cooled to -80 ° C for the next steps. These samples were quantified their RNA using
graphs (Nanodrop) to determine purity and absorbance ratio of the sample by the Beer-
Lambert law. In order to find the final samples on the microarray with markers, we
conducted a cDNA synthesis and labeling, preserving the RNA in ice cold
temperatures. This step was carried using the 3DNA Array 350Kit from Genisphere
company. Procedure involves the synthesis of cDNA using reverse transcriptase enzyme,
a mixture of deoxynucleotide triphosphate and an initiator of poly-thymidine triphosphate. 

A sequence of RNA-RT primer for each of the samples but add Cy3 (green) to

the control and Cy5 (red) to treatment of RA. Was necessary to carry out a pre-
wash the microarray slide is immersed in detergent and warm microarray several times to
avoid contamination by external agents that could affect the results. The process
carried out by the technicians was to hybridize the cDNA. For these
steps, the microarray should be pre-heated
to 60°C, while preparing the cDNAsolution in a 1.5mL tube with a dT blocker (2μl), some 
nuclease-free after (17μL) andenhanced 2X hybridization buffer. Resuspending the solution
with a pipette, solution was poured on the cDNA microarray and a cover slip was placed.
After this we proceeded to make a post wash. The last step was done in this work was
to add a florocromo through 3DNA molecule hybridizing cDNA. The added step is
the addition of a Dye Saver, which helps the florocromo not degrade quickly. This
chemical is very dangerous for health and must read the instructions on
the handling of chemical waste. All these are processes require skills of operation of
instruments in the laboratory, hazardous waste management and skills to
manage the microarray analysis software, Magic Tool. However, many of these
methods usually are highly qualified to get the results we seek, if done correctly. All
instruments were provided by the 4036 Biology Laboratory, University of Puerto Rico, Río
Piedras campus following the protocols available for each process.

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Preliminary Data

Celullar density formula

[ # of count cells X dilusion factor x10 4 ] = total cells/mL

[ # de numbered quadrants]

Table 1. Celullar Density

Cell culture Sub culture RA Sub culture control
(cells/mL) (cells/mL) (cells/mL)

0.7x106 2.24x104 4.7x104

Figure 1. RNA bands 28S and 18 S, UV image,

Left column shows RNA’s control density.
Right column shows RNA’s RA density.

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Preliminary Data:

Graph 1. Using Nanodrop, the graph shows the control group RNA concentration
respect to the absorbance.

Graph 2. Using Nanodrop, the graph shows a higher RNA concentration for the
treatment group respect to the absorbance compared to control.

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Preliminary data:

Table 2. Expressed genes chosen randomly from the WNT family genes
Tumor Supressor Proto-Oncogenes
Genes

Wnt2 Wnt1

Wnts5a Wnt7b

Wnt8a

Wnt8b

Table 3. Regulation on gene expression

Genes Expression Type of gene

Wnt2 2.230965 Tumor supressor

Wnt5a 2.231655 Tumor supressor

Wnt7b 0.239871 Proto-oncogene

Wnt1 0.130516 Proto-oncogene

Wnt8a 0.238143 Proto-oncogene

Wnt8b 0.106031 Proto-oncogene

<.45 significant
decrease
>3.0 significant
upregulation
=1.29 no regulation

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Preliminary Data:

Proto-Oncogenes
Wnt8b
Wnt8a
Wnt1
Wnt7b
Wnt2
Tumor Supressor Genes Wnt5a

0 0.5 1 1.5 2 2.5

Graph 3. Expression of WNT Genes in Cell Culture

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Discussion

The proliferation of B lymphocyte cells influenced by retinoic acid was the expected
behavior. The concentration of RNA in the Nanodrop graphs (Graphs 1 and 2, pag. 4)
indicated in a successful effect of retinoic acid in B cells, reviewed in literature and proven
by the hypothesis. As can be seen in Figure 3, p. 7 Wnt2 and Wnt5a genes were the genes
that showed a tendency to be up regulated. These genes have the function of activated
tumor cells suppressors. While the rest of the genes are downregulated. These four genes
are co-related with the activation of proto-oncogenes cells. Possible reason of change in
expression would be an interaction of RA between WNT gene products or direct interaction
with the WNT signaling pathway. RA treatment will increase expression of genes
associated with degradation of gene transactivation, while decreasing gene expression that
induced cell proliferation. Meanwhile, at the beginning of the study we see that the calculus
of sub culture density mismatch in the other tests. This could be a mistake in calculations or
counting with hemacytometer.You could say that the result have a significantly positive
trend because of the few Wnt family genes were chosen, much  was shown to be
downregulated to proto-oncogenes.  As we can understand the results, it is appropriate to
continue studies in WNT genes under the influence of retinoic acid in cancer
treatment. This would prove a key in new treatments for cancer. As an extension of this
study, we can carry out the experiment with the rest of the WNT genes that were not taken
into study, and assess what is the behavior of cells under the effect of treatment with
retinoic acid. Also worth studying specific receptors involved in each of these genes that
make them vary in features and how they affect the signaling pathway.

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