4. Results
4.5. In vivo assays
4.5.2 Hoxb4 BM transplantation experiments in mouse model
4.5.2.4 Overexpression of Hoxb4-∆Pro is associated with acute leukemia in transplanted
8-10 weeks old lethally irradiated (850 cGy) recipient mice (C3H-C57Bl/C3H-PeB) were injected with 1.13x105 to 1.8x107 retrovirally transduced 5-FU enriched BM progenitor cells. The recipient mice have been transplanted with
Fig. 4.5.2.3. Surface markers analysis of GFP+ WBCs from peripheral blood of m
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Chapter 4 - Results
progenitor cells expressing the following genes: Hoxb4-wt (n=5 (n=9), Hoxb4-∆Pro pre-treated for 1 week with VPA (n=3), Ho double mutant (n=3), Hoxb4-∆Pro-PBX pre-treated for 1 week w Hoxb4-∆Pro-HD (n=4) and GFP control (n=3) (Table 4.5.2.4). T
monitored for symptoms of hematological disorders, which included frizzled body hair, paleness in the extremities and lethargy. The examinatio
symptoms at sacrifice includes the measurement and the morpho of leukocytes/erythrocytes in the peripheral blood and in the bon well as th
), Hoxb4-∆Pro xb4-∆Pro-PBX ith VPA (n=3),
he mice were
n of leukemic logical analysis
e marrow, as e analysis of the spleen (weight and size). Various organs of leukemic ical
8.3 d) without for
these mice, with 0.5-1x106
animals. The 6.5 days post
died 42.8 days in case of a cy time of the secondary to suggests rexpression of multistep process. Few mice have been also injected with BM progenitor cells expressing Hoxb4-∆Pro pre-incubated with VPA for one week before transplantation. These mice died of leukemia 161 days (mean) after transplantation showing a prevalence of myeloid immature cells in the organs analyzed (84.6% of WBCs were expressing Mac-1), splenomegaly and leukocytosis (3.8x107 WBCs/ml). Apparently, the pre-treatment with VPA did not affect the development of the disease.
mice were fixed in formalin for histopathological and immunohistochem examinations.
All the Hoxb4-wt mice survived more than 404 days (mean 48 showing any hematopoietic malignancy. One out of 6 mice died associated illness.
The Hoxb4-∆Pro mice died 90-384 days after transplantation (mean 255 d, p<0.001 vs. Hoxb4-wt) by hematological malignancy. From
secondary syngeneic recipient mice (n=6) have been transplanted cells obtained from BM or spleen of diseased primary transplanted mean survival time of these secondary transplanted mice was 6
transplantation. The BM and spleen cells of these secondary transplanted mice have been used to transplant tertiary recipient mice (n=4), which
(mean) after transplantation for the same AML. As expected transplantable disease, we observed a shortening of the laten disease of 3.8fold from primary to secondary and of 1.5fold from tertiary serial transplantations of recipient animals (Fig. 4.5.2.4c). This that the disease in primary transplanted mice induced by the ove Hoxb4-∆Pro probably reflects a
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Chapter 4 - Results
All the mice transplanted with Hoxb4-∆Pro overexpressing BM p died of AML without maturation (Fig. 4.5.2.4a and Fig. 4.5.2.4 progression is often marked by the infiltration of blasts in variou therefore we assessed sacrificed moribund mice for these sign
histopathological sections of fixed organs followed by immunohistochem staining. In our model the diffuse neoplastic infiltration was involvin
liver, lymph nodes, lung, heart and kidneys. The infiltration of leuk the non-hematopoietic organs in general and to the brain in p striking, highlighting the aggressive nature of the disease. The type
of the cells involved in leukemia can be identified by performing immunohistochemical stainings. The myeloperoxidase distinguis
immature cells in acute myeloid leukemia (cell stain positive) and lymphoid leukemia (cells stain negative) (Fischbach 1996). Lymph characteristically negative for myeloperoxidase and chloracetate
rogenitor cells b). Leukemia s organs and s by making ical g the spleen, emic blasts to articular was and maturity
hes between those in acute
oid blasts are esterase. In order to determine the nature of the blasts, we performed various stainings of the organs from primary leukemic mice. Here we found that the neoplastic cells were myeloperoxidase (MPO) positive confirming the diagnosis of acute myeloid leukemia without maturation.
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Chapter 4 - Results
Fig. 4.5.2.4a. Histological analysis of multiple organs obtained from a represen mouse, transplanted with cells obtained from the infiltrated spleen of a diseased prim originally transplanted with Hoxb4-∆Pro expressing BM progenitor cells (Mous in Fig. 4.5.2.4b, the cells are large with abundant cytoplasm, blastic chroma
nucleolus. There is no myeloid differentiation. In the spleen there are some residual small areas of erythropoiesis. In the kidney the neoplastic
tative secondary ary mouse, e #33). As well as tin and prominent cells are identified in the capillary lumens in the glomeruli. The type of infiltration in the liver, lung and kidney clearly suggests that the mouse had a leukemic phase. The heart has a recent myocardial infarct with neoplastic cell infiltrating the myocardial wall. The residual white pulp of the spleen reveals cells that are CD3 and B220 positive but the tumor cells are negative for these markers.
Fig. 4.5.2.4b. Histological analysis of multiple organs obtained from a tertiary mouse, transplanted with cells obtained from the infiltrated BM of a secondary diseased mouse, originally transplanted with Hoxb4-∆Pro expressing BM progenitor cells (Mouse #36). For description, refer to legend Fig. 4.5.2.4a.
Effect of the additional PBX interacting domain (ID) and homeodomain (HD) mutations on the survival of Hoxb4-∆Pro leukemic mice. The combination of the PBX interacting domain mutation with the deletion of the proline-rich region has been investigated by transplanting lethally irradiated recipient mice (n=3) with BM
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Chapter 4 - Results
cells overexpressing the double mutated Hoxb4-∆Pro-PBX. These mice d leukemia 311 days (mean) after transplantation, showing no signif
in comparison to the Hoxb4-∆Pro mice. Moreover, BM cells expre
∆Pro-PBX have been pre-incubated with VPA (1mM) for
transplanted into three recipient animals. These mice died 338 days (mean, n=3) after transplantation. This “intermediate” survival rate of these two
was not significantly different when compared to the survival transplanted with HOXB4-wt and to the mice transplanted wit alone. Moreover, clinical features of these two groups of mice are
Hoxb4-∆Pro-PBX mice showed leukocytosis (1.2x107/ml), myeloid prevalence in the peripheral blood (81.32% Mac-1+ WBCs) and moderate splen
mg spleen
ied of icant difference
ssing Hoxb4-1 week, and
groups of mice of the mice h Hoxb4-∆Pro
of note. The
omegaly (300 weight), while the Hoxb4-∆Pro-PBX+VPA mice showed WBCs counts the peripheral
le 4.5.2.4 and
te ly, in transp d w o c verexpressing
Hoxb4-∆Pro-HD not ob a lo m e ent, suggesting that the stem cell activity as well as for the
ev t of t mia ob d in th odel.
(6x106/ml), spleen weight (200mg) and myeloid prevalence in blood (60.3% of WBCs) comparable to the GFP control mice (Tab Fig. 4.5.2.4c).
In resting the mice lante served
ith BM pr ng ter
genitor ngraftm
ells o we did
the interaction with the DNA is essential for
d elopmen he leuke serve is m
No. Mouse Retroviral
construct GFP+ injected
cells
Dead n days
after Tx Average d Notes (p vs wt)
1 4421#1 Hoxb4-wt 1.12x10 404 5
2 4421#2 Hoxb4-wt 1.12x105 416
3 4622 H #1 Hoxb4-wt 2x104 545
4 4622 H #2 Hoxb4-wt 2x105 483
5 4622 H#3 Hoxb4-wt 2x105 572
6 4622 B#4 xb4-wt 2x1 510
488.3
Ho 0 3
7 3970 A #2 Hoxb4-∆Pro 1.8x107 n.a.
8 3970 A #3 Hoxb4-∆Pro 1.8x107 279
9 3970 A #4 Hoxb4-∆Pro 1.8x107 126
10 3970 A #5 Hoxb4-∆Pro 1.8x107 279
11 3860 A#1 Hoxb4-∆Pro 6.3x105 n.a.
12 3860 A #2 Hoxb4-∆Pro 6.3x105 337
13 3860 B #1 Hoxb4-∆Pro 6.3x105 231
14 3860 B #2 Hoxb4-∆Pro 6.3x105 90
15 3860 B #4 Hoxb4-∆Pro 6.3x105
255 (p<0.001)
244
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Chapter 4 - Results
16 4622 I #1 Hoxb4-∆Pro 2x105 325
17 4622 I #3 Hoxb4-∆Pro 2x105 384
18 4622 F #3 Hoxb4-∆Pro 2x104 384
19 3970 B #5 Hoxb4-∆Pro-Pbx 1.7x107 153
20 3970 C#1 Hoxb4-∆Pro-Pbx 1.7x107 470
2 C Pro- 1.7
311 (p=0.080)
1 3970 #3 Hoxb4-∆ Pbx x107 310
22 3970 B #1 Hoxb4-∆Pro+VPA 2.5x107 154 23 3970 B #3 Hoxb4-∆Pro+VPA 2.5x107 156 24 3970 B #4 Hoxb4-∆Pro+VPA 2.5x107 174
161 (p<0.0001)
25 3970 C #4 b4-∆Pr
x+VPA 1.8
Hox
Pb o- x107 416
26 3970 D #1 b4-∆Pro
x+VPA 1.8x 7 4
Hox
-Pb 10 19
27 3970 D #2 b4-∆Pro
VPA 1.8x
338 0.083) (p=
Hox
-Pbx+ 107 405
28 4614 B #2 Hoxb4-∆Pro 1x10 6 50 2nd Tx
29 4614 C #1 Hoxb4-∆Pro 1x106 54 2nd Tx
30 4614 C #2 Hoxb4-∆Pro 1x106 54 2nd Tx
31 4614 C #3 Hoxb4-∆Pro 1x106 54 2nd Tx
32 4614 C #4 Hoxb4-∆Pro 1x106 82 2nd Tx
33 4614 C #5 Hoxb4-∆Pro 1x106 105
66.5 (p<0.0005
. 1st Tx) vs
2nd Tx
34 4685 #1 Hoxb4-∆Pro 1.2x10 51 6 3rd Tx
35 4685 #2 Hoxb4-∆Pro 1.2x106 42 3rd Tx
36 4685 #3 Hoxb4-∆Pro 1.2x106 43 3rd Tx
37 4685 #4 b4-∆Pr 35
43 (p<0.0013
vs. 1st Tx)
3rd Tx
Hox o 9x106
38 5264 #2 Hoxb4-∆Pro-HD 2.2x10 5 321 *
39 5331 #1 Hoxb4-∆Pro-HD 3x105 >331 *
40 5331 #2 Hoxb4-∆Pro-HD 3x105 >331
327
*
GFP 3.7x105 >400
41 5020
42 5029 GFP 1.3x106 >400
43 4951 GFP 1.5x106 >400
Table 4.5.2.4. List of transplanted mice. All the #3970 mice have been injected with cells which have been cultivated for 1 week after transduction, with or without VPA. For all the mice a number of 5-FU enriched GFP- progenitor carrier cells have been injected (3x105-2x106). The mice injected with higher dilutions of BM cells for estimation of the CRU frequencies are not reported. *
= these animals did not show engraftment.
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Chapter 4 - Results
mice. The animals which died ncluded.
a
Fig. 4.5.2.4. Kaplan-Meier survival curves of Hoxb4 transplanted shortly after transplantation by irradiation related mortality are not i
4.5.2.5 Clinical features of the Hoxb4-∆Pro associated leukemi