Post on 06-Apr-2018
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UNIVERSIDAD COMPLUTENSE DE MADRID,
(Spain)
FACULTAD DE MEDICINAFACULTAD DE MEDICINA
- new relevant biomolecular concept, its
applications in cancer research andother research fields
A. Ferreira-Alemo, MD PhD
RIBOGRAMA:
New Pathways inCancer Research
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The purpose of this research, which was presented as a Doctoral Thesis, at
UNIVERSIDAD COMPLUTENSE DE MADRID is draft a method of:
- diagnosis,
- screening and
- monitoring
of the colon and rectum cancer, founded on technical and current concepts of Molecular Biology
This new method allows a diagnosis in a "pre-carcinoma in situ stage,
since it is based on the phenotype of mucosal cells, composed ofvery high
quantities of free ribosomes in the cytoplasm of colonocytes, which can be
quantified by means of the of the flow cytometry, after its isolation, and
labelled with fluorochromes.
The repeated records of those quantities of free ribosomes, establishes a
graphic curve (RIBOGRAMA) that represents the degree of a malignanttendency, which above a certain level of concentration, allows to say that the
cells in a tissue (of the colon and rectum, e.g.) can be considered in the process
of developing carcinoma in the colon-rectum mucosa, before any macroscopic
viewing (endoscopy).
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biomolecular basis of the cellular machinery
FREE AND MEMBRANE RIBOSOMES
Free ribosomes are generally assumed to synthesize intracellularproteins (internal
economy of the cell), whereas bound ribosomes synthesize proteins intended for
secretion (exportation).
DNA
RNA
CELLULARNUCLEUS CYTOPLASM
ROUGHENDOPLASMICRETICULUM
FREERIBOSOMES
MEMBRANE
RIBOSOMES
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Schematic three-dimensional rough endoplasmic reticulum, showing the synthesis of
proteins destined for export by ribosomes attached to membranes reticulum
(signal sequence).
biomolecular basis of the cellular machinery
MEMBRANE RIBOSOMES
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RIBOSOME
TumorMarkers
U p s t r e a m s i d e Downstream side
The protein synthetic capacity of a cell is
dictated by a number ofprocesses such:
biomolecular basis of the cellular machinery
CONTROL OF PROTEIN SYNTHESIS
mRNA availability; efficiency of translation; availability of translation factors;
number of ribosomes
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biomolecular basis of the cellular machinery
THE RIBOSOME AS THE COMMON ELEMENT IN THE
CELLULAR BIOMOLECULAR PROCESS
1. The evidence accumulated to date
indicates that overall the protein syntheticcapacity is determined by the number of
free ribosomes, which function as an
informative sensorof the protein synthesis.
2. Increased cell proliferation is a hallmark
of agressive malignant neoplasms, whichrequires a general increase in protein
synthesis and a specific increase in the
synthesis of replication-promoting proteins.
3. Ribosome synthesis, or biogenesis, is a complex process dependent on the
coordinated synthesis of approximately 85 ribosomal proteins, four ribosomal RNA(rRNA) and their subsequent processing and assembly into mature ribosomes.
(Tumor Markers)
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It has been shown in polyoma virus-transformed cells that one of the key mechanisms
for the loss of control ofprotein synthesis is an inability to decrease ribosome number,
which correlated with proliferation of transformed cells in fresh media without addition
of serum growth factors (Stanners et al., 1979).
CELL CYCLE
Hours Hours
Minutes
FREERIBOSOMES
QUANTITY
INTERPHASE MITOSIS INTERPHASE MITOSIS
G1 S G2 M G1 S G2 M
9,3 8,0 2,0 0,7 9,3 8,0 2,0 0,7
PRO MET ANA TEL
25,2 2,1 2,1 12,6
NORMAL CELL
NORMAL CELL
HYPERPLASIACELL
HYPERPLASIACELL
MALIGNANTTRANSFORMED
CELL
biomolecular basis of the cellular machinery
THE RIBOSOME AS THE COMMON ELEMENT IN THE
CELLULAR BIOMOLECULAR PROCESS
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biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
- From the subjective nature, relatively of the amount offree ribosomes,
a diagnosis of malignancy is made just under the idea of the constancy
property that consists of an great increase offree ribosomes in cells that
are in a process of multiplication without self control, as in the case ofmalignant cells;
- A malignant cell has a very high free ribosomes density that is a
fundamental aspect of its phenotype;
- So, naturally, to describe the quantities of free ribosomes it is
necessary to create a language reproducible, not subjective, with
mathematical translation, through a graphic curve that we callRIBOGRAMA.
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A perspective on the biology of malignant cells
Several lines of evidence, some well
established and recent, sayemphatically the idea that RNA
content is high in cancer cells and
genetic events that lead to cancer
are often linked directly or indirectly
to the ribosome biogenesis.
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
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RIBOGRAMAGRAPHICCURVE
Legend:
smt = every 6 months; A, B, C, D =Concentration levels of free ribosomes
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
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In the decades of 60s and 70s were
published studies concerning the
accumulation of free ribosomes duringchemical induction of neoplastic growth,
through initiation by 7,12-
dimethylbenz(a)anthracene and promotion
caused by 12-O-tetradecanoyl-phorbol-13-
acetate, in the interfollicular areas of the
dorsal skin of mice.
Accumulation of ribosomes in the process of oncogenesis
biomolecular basis of the cellular machinery
RIBOGRAM CONCEPT
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Case Number Quantitative description of free
ribosomes in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / Information References
(Publishedi n the Doctoral Thesis)
1 Numerous free ribosomes Citoplasma filled numerous
organelos, mitochondrias, free
ribosomes, short
Carcinoma pappilar invasivo,
moderadamente diferenciado
396
2 Free ribosomes randomly dispersed Small clusters of free ribosomes Glial C6 Cells transformadas 397
3 Free ribosomes excedingly abundant --------------------- Human adrenocortical carcinoma
derived SW-13 cell lines
398
4 Numerous free ribosomes Few granular endoplasmic reticulum Human osteosarcoma 399
5 Increased free ribosomes Membrane bound ribosomes
decreased
Liver tumor rats 400
6 Free ribosomes Few organelles Hepatoma cell lines 401
7 Numerous free ribosomes and
polysomes
Some rough endoplasmatic
reticulum
EBV Related liver tumor (occurring
after kidney transplant)
402
8 Increase of free ribosome numbers --------------------- HEL cells human erytroleukemiainduced
403
9 Principal cytoplasmic organelles were
free ribosomes into rosettes
Membranes of rough endoplasmic
reticulum and Golgi apparatus were
poorly developed in most neoplastic
cell
Bronchogenic carcinoma 404
10 Rich in ...free ribosomes Rich in organelles of this type
including microtubules
mitochondria
Oligodendroglioma 405
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free
ribosomes in EM (Electrom Microscope)
Electrom Microscope Main General View Diagnosis / Information References
(Publishedi n the Doctoral Thesis)
19 exhibited greater activity (free
ribosomes)
Hepatoma transplantable 414
20 Turnover of free ribosomes was
greater in host livers
Transplantable hepatoma rat cells 415
21 Free ribosomes were the most prominent
cellular organelles
Moderate number of mitochondria, were
randomly distributed. Poorly developed
granular endoplasmatic reticulum
Nasofaringeal carcinoma 416
22 Disagregation of free and membrane
bound polyribosomes
---------------------- Effect of aflatoxin B1 on hepatic
polyribosomes and protein sintesis
in the rat
417
23 Free ribosomes Few organelles, small number of
mitochondria, poor developed Golgi
complexes inconspicuous rough
endoplasmic reticulum
Keratin positive Ewings sarcoma 418
24 Prominent ultrastructural features
being free ribosomes
Prominent
- rough endoplasmic reticulum cisternal -
mitochondria
Human adrenocortical carcinoma
derived SW-13 cell line
419
25 Prominent free ribosomes Prominent rough endoplasmic reticulum Thalamic tumor in a patient with
human T-cell lymphotropic virus
type 1 (HTLV-1) [extranodal
lymphoma of the skull]
420
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free
ribosomes in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / Information References
(Published in the Doctoral Thesis)
11 Numerous free ribosomes Enlarged mitochondria prominente
smooth and rough endoplasmic
reticulum
Mammary Paget Disease 406
12 Numerous free ribosomes ----------------------- Intestinal tumor induced by 1,2 -
dimethylhydrazine
407
13 Increase in membrane free ribosomes Functional alterations paralleled by
an increase in membrane free
ribosomes
Rat liver hepatocarcinogenesis
induced by 0,25% DL ethiomine
[hepatoma]
408
14 cytoplasmicmainly on free ribosomes Weakly rough surface endoplasmic
reticula cisternae and Golgi
complexes
Neuroblastoma cell lines 409
15 After 2 to 3 weeks ofATRA treatment
ER and free ribosomes became rare as
the maturation process
Acute promielocytic leukaemia cells 410
16 Cytoplasm may contain free ribosomes Few mitochondria, a dilated rough
endoplasmic reticulumMetastatic (neck lymph node)malignant extrarenal rhabdoid tumor
411
17 predominantly free ribosomes Inconspicuous rough
-endoplasmic reticulum
-small number of mitochondria
-developed Golgi complexes
Ewings sarcoma 412
18 Free ribosomes w ere present Mitochondria endoplasmic
reticulum and Golgi apparatus were
present
Colorectal polyps 413
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free ribosomes
in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / Information References
(Published in the Doctoral Thesis)
26 Accumulation of numerous free ribosomes Marcadly increased vesicular
trafficking the Golgi
Neuroblastoma B-104 cells 421
27 Free ribosomes Cytoplasmic organelles such as
rough endoplasmic reticulum,
mitochondria
Carcinoma of the thyroid gland 422
28 Numerous free ribosomes Abundance of dilated rough
endoplasmic reticulum
Small cell carcinoma of the ovary 423
29 Abundant free ribosomes -Absent smooth endoplasmic
reticulum
Carcinoma of the endometrium 424
30 Abundant free ribosomes Abundant rough endoplasmic
reticulum cisternae
Smooth endoplasmic reticulumabsent
Adrenocortical oncocytoma 425
31 Abundant free ribosomes Prominent nucleoli Chemically induced mouse
hepatoblastoma
426
32 Numerous free ribosomes Small amounts of rough
endoplasmic reticulum and round
mitochondria
Malignant fibrous histiocytoma 427
33 Numerous (?) free ribosomes Numerous small mitochondria Adenocarcinoma cells of an ovarian
clear cell tumor
428
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free ribosomes
in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / Information References
(Published in the Doctoral Thesis)
34 Increase of free ribosomes Prominent nucleoli, marked decrease
in rough endoplasmic reticulum;
abundant microphilaments and
microtubules
Histogenesis ofpseudoductular
changes in pancreas of guinea pigs
with N-methyl, N-Nitrosourea
429
35 Many free ribosomes Large nuclei with prominent nucleoli;
small quantities of rough and smooth
endoplasmic reticulum
NCI H295R cells 430
36 Free ribosomes were common -Rich mitochondria
- Some rough endoplasmic reticulum
Leiomyoblastoma of the uterus 431
37 Increased free ribosomes Membrane bound ribosomes
decreased
Liver tumor rats 432
38 High content s o f free ribosomes ----------------------- Sarcoma 45 433
39 Clusters of free ribosomes Dilated endoplasmic reticulum
profiles
Cortical dendritic spines 434
40 Many free ribosomes Many rough endoplasmic reticuli Pulmonary clear cell carcinoma 435
41 Numerous free ribosomes ----------------------- Hepatocarcinogenesis induced by
ethionine
436
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free
ribosomes in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / Information References
(Published in the Doctoral Thesis)
42 ...Contain a few cytoplasmic organelles
...except for free ribosomes
Contain few cytoplasmic organelles
and a small number of
mitochondria
Human germ cell tumor derived cell
line
437
43 tumor cells contains free ribosomes
contain filamentous structures Embryonal rhabdomyosarcoma in bucal
mucosa
438
44 Abundant free ribosomes Abundant mitochondria Oncocytic metaplasia and carcinoma of
the endometrium
439
45 referncia a free r ibosomes ...Referncia a
-Golgi complex
-Mithocondria
-RER
Tapioca melanoma of the iris... 440
46 free ribosomes w ere present Rough endoplasmic reticulum were
present
lipomatous lesions in the liver of
B6C3F1 mice
441
47 numerous free ribosomes numerous distended rough
endoplasmic reticula, Golgi
apparatus
Pituitary fibrosarcoma following
radiotherapy
442
48 Abundant free ribosomes Abundant mitochondria, lysosomes,
rough surface endoplasmic
reticulum
Epithelioid malignant schwannoma cell
line
443
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205))
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Case
Number
Quantitative description of free ribosomes
in EM (Electrom Microscope)
Electrom Microscope Main General
View
Diagnosis / InformationReferences
(Published in the Doctoral Thesis)
49 free ribosomes were observed desmosomes present Rat bladder tumor cell lines 444
50 increases of free ribosomes Nuclear lobulation and nucleolar
content
Human erythroleukemia cells 445
51 Numerous free ribosomes Some mithocondria and other
poorly developed cytoplasmic
organelles, suggesting
undifferentiated nature
Rat malignant fibrous histiocytoma 446
52 Many free ribosomes ----------------------- Rat colorectal epithelium tumors after
treatment with carcinogen
447
53 Numerous free ribosomes Poorly differentiated cytoplasm
numerous mitochondria
Small cell osteosarcoma 448
54 Abundance of free ribosomes Paucity of organelles Ewings sarcoma 449
55 Numerous free ribosomes Little rough endoplasmic
reticulum; few mitochondria; small
Golgi complex
Mouse mammary gland
adenocarcinomas
450
56 Great number of free ribosomes ----------------------- Malignant lymphoma 451
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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Case
Number
Quantitative description of free ribosomes in
EM (Electrom Microscope)
Electrom Microscope Main General View Diagnosis / InformationReferences
(Published in the Doctoral Thesis)
57 Large amount of free ribosomes Well developed Golgi apparatus, rough
endoplasmic reticula
Monocytic leukaemia cells 452
58 Free ribosomes Roughendoplasmic reticulum
pleomorphic mitochondria
Poorly differentiated Rhabdomyosarcomas 453
59 805 free ribosomes ----------------------- Lymphocytic leukaemia tumor 454
60 Clusters of free ribosomes ProminentGolgi apparatus numerous
profiles of rough endoplasmic reticulum
Adenocarcinoma from apocrine glands in dogs 455
61 Few free ribosomes Poor developed Golgi apparatus
phenotypic features characteristics for
myeloma cells
Myeloma cells in human hybridoma 456
62 Numerous free ribosomes Few granular endoplasmic reticulum Human osteosarcoma 457
63 Many free ribosomes Numerous organelles many
lysosomes, swollen mithocondria, Golgi
complexes, rough and smooth
endoplasmic reticulum
Subependymal giant cell tumor 458
biomolecular basis of the cellular machinery
TABLE I - QUANTITATIVE DESCRIPTION OF FREE RIBOSOMES ON E.M. OBSERVATION
(FIRST 63 CASES AMONG A TOTAL OF 205)
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biomolecular basis of the cellular machinery
TABLE II - DIFFERENT WAYS HOW IS DESCRIBED THE CONTENT OF FREE RIBOSOMES IN
ATYPICAL CELLS, ON E.M. - ELECTRON MICROSCOPE (example of the ten cases of table i)
N de caso Descripcin cuantitativa de los ribosomas
libres
1 Numerous free ribosomes
2 Free ribosomes randomly dispersed
3 Free ribosomes excedingly abundant4 Numerous free ribosomes
5 Increased free ribosomes
6 Free ribosomes
7 Numerous free ribosomes and polysomes
8 Increase of free ribosome numbers
9 Principal cytoplasmic organelles were freeribosomes into rosettes
10 Rich in ...free ribosomes
Biomolecular analysis of malignant cells
Content of free ribosomes in malignant cells
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biomolecular basis of the cellular machinery
TABLE III-A - (NUMEROUS FREE RIBOSOMES)
Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
1 Papilar Invasive Carcinoma 50 Small cell osteosarcoma
9 Mammary Paget disease 52Mouse mammary gland
adenocarcinoma
10 Intestinal Tumor 59 Human osteosarcoma
24 Neuroblastoma 75 Testicular seminoma
26 Small cell carcinoma of the ovary 87 Phylloides tumor of the prostate
30 Malignant fibrous histiocytoma 88 Malignant Choroid Plexus papiloma
31 Adenocarcinoma of ovary clear cell 91 Malignant fibrous histiocytoma
38Hepatocarcinogenesis induced by
ethionine105 Small cell carcinoma of ovary
44 Pituitary fibrosarcoma 107 Mammary Paget disease
48 Rat malignant fibrous histiocytoma 108 Adenocarcinoma of stomach
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Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
117 Small cell carcinoma of the ovary 142 Testicular seminoma
118 Neuroblastoma 151Mouse mamary gland
adenocarcinoma
123Rat hepatocarcinogenesis induced
by ethionine153 Osteosarcoma
125 Small cell osteosarcoma 163 Mt TW15 mamosotrofic tumor
127Transplantable Rat malignant
fibrous histiocytoma178
Hyperplasic foci in precancerous
rat liver
129 Pituitary adenoma 191 Human tongue carcinoma cells
131 Large malignant choroid plexus 193 Intestinal tumors
132 Control neurocitoma
LEGEND:Sample of cases 37Phenotypes of malignancy observed 35
PERCENTAGE OF MALIGNANT TISSUES - 95%
biomolecular basis of the cellular machinery
TABLE III-A - (NUMEROUS FREE RIBOSOMES)
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biomolecular basis of the cellular machinery
TABLE III-B - (RICH IN FREE RIBOSOMES)
Case Number Diagnosis/ Information
8 Oligodendroglioma
96 Adenocarcinoma of the stomach
106 Oligodendroglioma
140 Naked nuclei in breast aspirate smears
197 Epidermal carcinogenesis
LEGEND:Sample of cases 5Phenotypes of malignancy observed 5
PERCENTAGE OF MALIGNANT TISSUES - 100%
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biomolecular basis of the cellular machinery
TABLE III-C - (ABUNDANT FREE RIBOSOMES)
Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
3 Adrenocortical carcinoma 90Chemically induced mouse
hepatoblastoma
27 Carcinoma of the endometrium 95 Adrenocortical carcinoma
28 Adrenocortical oncocytoma 98 Carcinoma of thyroid gland
29Chemical induced mouse
hepatoblastoma104 Ductal papillary adenocarcinoma
41 Carcinoma do endometrium 115 Carcinoma of endometrium
45
Epithelial malignant schwannoma
cell line 116 Adrenocortical oncocytoma
51 Ewings sarcoma 128Epithelioid malignant schwannoma
cell lise
69 Brain tumor in doges 138 Mouse hepatoblastoma
71 Vaginal hemangiopericytoma 145 Vaginal hemangiopericytoma
80 Mouse hepatoblastoma 146 Brain tumor
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Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
156 Extra skeletal Ewings sarcoma 176 Adrenal cortical tumor
160 Neuroblastoma 183 Pseudolymphoma of the lung
165 Epithelial tumors induced in ratkidney
187 Salivary gland carcinoma
166 Hepatoblastoma of foetal liver 188 Epithelioid sarcoma
168 Hepatocellular tumors 189Malignant epithelioid
schwannoma
171 Stromal sarcoma of breast 199Primary lymphosarcoma of
testis
173 Cholangiocarcinoma 201 Ependymoma
LEGEND:Sample of cases 36Phenotypes of malignancy observed 34
PERCENTAGE OF MALIGNANT TISSUES - 94,4%
biomolecular basis of the cellular machinery
TABLE III-C - (ABUNDANT FREE RIBOSOMES)
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biomolecular basis of the cellular machinery
TABLE III-D - (MANY FREE RIBOSOMES)
Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
33 NCI-H295R cells 124 Pulmonary clear all carcinoma
37 Pulmonary clear cell carcinoma 126 Colorectal tumors
49 Colorectal epithelium tumors130 Human neuroblastoma cell line
60 Subependymal giant cell tumor 137 Malignant fibrous histiocytoma
73 Pancreatic islet cell tumor 143 Pancreatic islet cell tumor
89 Neuroblastoma cell line 155 Malignant lymphoma
101 Primary osteoblast like cells 164 Spontaneous testicular neoplasm
102 Human pituitary tumor 196
Epidermis experimental
carcinogenesis
112NCI-H295R cells (human adrenal
cell lines)202 Malignant lymphoma
122Pluripotent human germ cell tumor
derivated cell lineLEGEND:Sample of cases 19Phenotypes of malignancy observed 17
PERCENTAGE OF MALIGNANT TISSUES - 89,4%
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biomolecular basis of the cellular machinery
TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)
Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
6 Human erythroleukemia cells 62 Papillary thyroid carcinoma
11 Rat liver hepatocarcinoma induced 77 Medullary carcinoma of the thyroid
15 Ewings sarcoma 79 Cutaneous neurofibromas
19 Nasofaringeal carcinoma 84 R and T head and neck carcinomas
22 Adrenocortical carcinoma 100 Human erythroleukemia cells
23 Thalamic tumor 109 Transplantable hepatomas
32Pseudoductular changes in pancreas
pigs110
Adrenocortical carcinoma
34 Leiomyoblastoma of the uteros 113Pseudo-ductular changes in
pancreas of pigs
35 Liver tumor rats 120 Embryonal Rhabdomyosarcoma
47 Human erythroleukemia cells 133Seminoma and parathyroid
adenoma
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Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
141 Medullary carcinoma 181Malignant fibrous histiocytoma of
the orbit
157 Oesophageal epithelium dysplastic
foci of the thyroid185 Clear cell thyroid carcinoma
159 Pituitary adenomas 186 Hepatocarcinoma
167 Uveal malignant melanoma 204Carcinogenesis induced by 4-
dimethylaminobenzene
LEGEND:Sample of cases 38Phenotypes of malignancy observed 35
PERCENTAGE OF MALIGNANT TISSUES - 92,2%
biomolecular basis of the cellular machinery
TABLE III-E - (INCREASED NUMBER OF FREE RIBOSOMES)
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biomolecular basis of the cellular machinery
TABLE III-F - (FREE RIBOSOMES)
Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
2 Glial C6 cells transformed 39Human germ cell tumor derived cell
line
5Administration of ethionine female
rats40 Embryonal rhabdomyosarcoma
7 Bronchogenic carcinoma 42 Tapioca melanoma of the iris
12 Neuroblastoma cell lines 43Lipomatous lesions in the liver of
B6 C3 F1 mice
14Malignant extra renal rhabdoid
tumor46 Rat bladder tumor cell lines
16 Colorectal polyps 55 Poorly differentiated
rhabdomyosarcoma
17 Hepatoma transplantable 56 Lymphocytic leukaemia tumor
21 Ewings sarcoma 57Adenocarcinoma from aprocrine
glands in dogs
25 Carcinoma of thyroid gland 63 Carcinoma of lung
36 Cortical dendritic spines 64 Friend erythroleukemia cell growth
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Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
65 Hodgkins disease 82 Rat pheochromocytoma cell
66 Hepatoma cells 83 Carcinoma of endometrium
67 Hepatoma cells 85 Myelogenous leukaemia cell line
68 Astroblastoma 86Seminoma and parathyroid
adenoma
70 Endometrial adenocarcinoma 92 Myeloma cells
72 Clear odontogenic tumor 94 Congenital fibrosarcoma
74 Cell condrosarcoma 97 Human tumoral ependymal celllines
76 Sphenoidol ridge meningioma 99Ductal adenocarcinoma of the
pancreas
78 Osteosarcoma cell line 103 Carcinoma rat testis
81 Malignant fibrous histiocytoma 111 Human adrenal cell lines
biomolecular basis of the cellular machinery
TABLE III-F - (FREE RIBOSOMES)
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Case
NumberDiagnosis/Information
Case
NumberDiagnosis/Information
119 Cerebral small cell tumor 150 Carcinoma of the lung
120 Embryonic rhabdomyosarcoma 154Adenocarcinomas (in dogs),
aprocrine glands
121 Nevic corpuscle 158 Branchiometric paragangliomes
134 Myelogenous leukaemia cell line 161 Adrenocortical carcinomas
135 Carcinoma of the endometrium 162 Melanocytomas of the optic nerve
136Adrenal chromaffin and
pheochromocytoma169 Transitional cloagenic carcinomas
139
N-methyl-N-amilnitrosamina
induced rat oesophageal
carcinogenesis
170 Maxilofacial synovial sarcoma
144 Clear odontogenic tumor 174 Cultured hepatoma cells
148 Rat ascitis hepatoma cells 175 Monoclonal Hodgkin cells cultured
149 Rat ascitis hepatoma cells 177 Gastric leiomyosarcoma
biomolecular basis of the cellular machinery
TABLE III-F - (FREE RIBOSOMES)
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Case Number Diagnosis/Information
180Establishment of a human rectal cancer
cell line
182 Pituitary adenomas
184 Myeloma cells
195Adenocarcinoma of the human tuba
uterina
198 Pituitary thyrotrophic tumor
200 Dermatofibroma (histiocytoma)
205 Friend erythroleukemia cell growth
LEGEND:Sample of cases- 70Phenotypes of malignancy observed 67
PERCENTAGE OF MALIGNANT TISSUES - 95,7%
biomolecular basis of the cellular machinery
TABLE III-F - (FREE RIBOSOMES)
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Table of
"groups of expressions"
Number of cases
in each group of
expressions
Malignant phenotypes in
each "group of
expressions"
%Malignant
tissues
I - (NUMEROUS FREE RIBOSOMES) 37 35 95 %
II (RICH IN FREE RIBOSOMES) 5 5 100 %
III (ABUNDANT OF FREE RIBOSOMES) 36 34 94,4 %
IV (MANY FREE RIBOSOMES) 19 17 89,4 %
V (INCREASED NUMBER OF FREE
RIBOSOMES)38 35 92,2 %
VI (FREE RIBOSOMES) 70 67 95,7 %
biomolecular basis of the cellular machinery
TABLE OF "GROUPS OF EXPRESSIONS" RELATIVELY
TO DESCRIBE THE CONTENT OF FREE RIBOSOMES
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biomolecular basis of the cellular machinery
THE RIBOSOME NUMBER AS A QUANTITATIVE PARAMETER
OF THE MALIGNANT PHENOTYPE TREND
1. The RIBOGRAME graphic curve reflects a dynamic idea of
changes in numerical values or quantities of all the free ribosomes.
2. The density offree ribosomes is the trend of the phenotype of the
malignancy of a cell, relatively to a normal range, of a community of
cells under observation in regard to biomolecular behavior,
representing a malignant growth of the cell phenotype, above a
certain level of increase.
3. It will be very important produce predictive mechanistic models
based on tumor dynamics, reflecting the translation in the cell
phenotype, for example (the count of free ribosomes) in a cell as
the phenotypic alteration process under the oncogenic stimulus.
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THEORETICAL CONSIDERATIONS ABOUT THE GRAPH
CURVE OF RIBOGRAMA AND ITS IMPLICATIONS IN THE
CLINICAL PRACTICE AND OTHER RESEARCH FIELDS
Legend:
smt = every 6 months; A, B, C, D =Concentration levels of free ribosomes
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THEORETICAL CONSIDERATIONS ABOUT THE GRAPH
CURVE OF RIBOGRAMA AND ITS IMPLICATIONS IN THE
CLINICAL PRACTICE AND OTHER RESEARCH FIELDS
INTERPRETATION OF THE GRAPH IC CURVE (RIBOGRAMA)
Curve that starts at the normal level and reaching the increased level,
which involves care for a prophylactic study on the reasons or causes that determine itsappearance. The cells at this level (increased) still have no security features of malignancy, but
their increased number of ribosomes continues growing. The cell is transformed into a
malignant phenotype (alarm level). At this point it is essential to make a dietary study to survey
and identify possible mutations responsible for the uncontrolled growth of ribosome biogenesis
in the process of carcinogenesis;
Segment of the curve derived from the anterior (segment a-b), which
already corresponds to the phenotypic alterations seen in electron microscopy, in which cells
are already present with phenotypic features of malignancy (significant increase in median
density of free ribosomes), which is the alarm level
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THEORETICAL CONSIDERATIONS ABOUT THE GRAPH
CURVE OF RIBOGRAMA AND ITS IMPLICATIONS IN THE
CLINICAL PRACTICE AND OTHER RESEARCH FIELDS
Segment of the curve, continuing the anterior segment, which represents the level of
signs and symptoms of the CRC disease (clinical level);
This segment of the curve corresponds to a change in direction of the curve a-b that
results from preventive action and treatment (medications and special diet)
Corresponds to a curve in which cells grow (multiply or proliferate) in a self-proliferation
mechanisms increased, as in the case of the seminiferous tubules, or the endometrium. This increased level of
ribosome biogenesis control is thereof within the physiological (non-malignant);
Normal curve
This work is intended to study the item colorectal cancer (CRC), in which ribogramacurve is high, so it is not the time to talk about the meaning of it at low level. Still, in this level, it will be useful tostudy conditions of degeneration and involution of cells and tissues, either the intestinal mucosa, or other extra-
intestinal tissue.
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CHARACTERISTICS OF MALIGNANCY VERSUS THE
COUNTING OF FREE RIBOSOMES
While Ribograma method gives a quantity idea of the cell phenotipic view, the descriptions
of Electron Microscope (EM) view of the cells with phenotypic characteristics of malignancy vary
by greater or lesser degree of differentiation of its texture or morphology, but these descriptions are
subjective, depending on the observer.
In terms of molecular biology of cancer, the malignant transformation of the normal cells is the
acquisition of a series of progressive specific genetic changes that act disobeying to the strong
antitumor mechanisms that exist in all normal cells, which include: a) - regulation of signal
transduction, b) - differentiation, c) - apoptosis, d) - DNA repair; e) - cell cycle progression, f) -
angiogenesis, g) - cell adhesion, which are not quantifiable, and in its whole do not allow a
monitorization reproducible and practical .
As in the descriptions of EM, these mechanisms of malignant transformation cannot be evaluated
by mathematic criteria, because the described features cannot be quantified. Similarly, the properties
of malignant transformed cells, growing in cell culture or in vivo, as observed by techniques of cell
biology or molecular biology do not allow a rigorous quantitative assessment, because they
depend on subjective criteria, without concrete numbers or standardized numerical limits.
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CHARACTERISTICS OF MALIGNANCY VERSUS THE
COUNTING OF FREE RIBOSOMES
A. The characteristics listed in next table are properties of malignant transformed cells
growing in cell culture (or in vivo), that are not susceptible of being quantified, while with
the quantification free ribosomes it is possible to make comparisons, through counting
them with the use offlow cytometry techniques. Then, with the count of free ribosomes ispossible to compare the results with reference to the time variable, like it is done with theRIBOGRAMA concept.
B. Of the characteristics listed in the next table it is not possible to register a numeric result,
because they do not show a mathematic and reproducible dynamic trend growth andproliferation status of a set of cells belonging to a given tissue, but
C. This is possible with the RIBOGRAMA concept.
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PROPERTIES OF GROWING MALIGNANT TRANSFORMED
CELLS IN A CULTURE OF CELLS AND /OR IN VIVO
1. Cytological changes similar to cancer cells in vivo, including increased cytoplasmic basophilia, number and size of the nuclei increased, increased nucleus-
cytoplasm relation, formation of clusters and cords of cells;
2. Altered growth characteristics;
a. "Immortality" of the transformed cells in culture. Transformed malignant cells become "immortal" in that they can be transferred in culture indefinitely;
b. Decreased dependent inhibition of cell density or loss of "contact inhibition ." The transformed cells often grow to a density higher than their normal
counterparts, and they can pile up in culture rather than stop growing when they contact each other;
c. Decreased serum needs. The transformed cells require decreased concentrations of serum or growth factors to replicate in culture compared to cells not
transformed.
d. Loss of anchorage dependence and acquisition of the ability to grow in soft agar. The transformed cells may lose their need to grow attached to surfaces and
can grow as colonies in semisolid free.
e.Loss of cell cycle control
. The transformed cells do not stop in G
1, or borderline G
1 /S cell cycle when subjected to metabolic constraints of growth
.
f. Resistance to apoptosis (programmed cell death).
3. Changes in the structure and function of the cell membrane - including increased agglutination by plant lectins, altered composition of cell surface glycoproteins,
proteoglycans, glycolipids and mucins; appearance of tumor-associated antigens, and increased uptake of amino acids, hexoses and nucleotides.
4. Loss of cell-cell and cell-extracellular matrix that promotes cell differentiation.
5. Loss of response to inducers of differentiation and altered cellular receptors for these agents .
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PROPERTIES OF GROWING MALIGNANT TRANSFORMED
CELLS IN A CULTURE OF CELLS AND /OR IN VIVO
6. Alteration of signal transduction mechanisms, including growth receptors constitutive phosphorylation cascades and mechanisms of dephosphorylation rather
than a regulated function.
7. Increased expression of oncogenic proteins due to translocation, amplification and chromosome mutation.
8. Loss of protein products of tumor suppressor genes due to deletion or mutation .
9. Genomic misreading that causes the overproduction of growth-promoting substances, eg, IGF-2.
10. Increase, or unruly production, of growth factors, e g, TGF-alpha, tumor angiogenesis factor, PDGF, hematopoietic growth factors (e g, CSFs, interleukins).
11. Genetic instability, leading to progressive loss of regulated cell proliferation, enhanced invasion and increased metastatic potential . Genes "mutator" may be
involved in this effect.
12. Altered enzyme profifes. The transformed cells have increased levels of enzymes involved in the synthesis of nucleic acids and produce higher levels of lytic
enzymes, e g, proteases, collagenases and glycosidases.
13. Production of oncodeveloping gene products. Many malignant transformed cells growing in culture or in vivo produce increased quantities of oncofetal antigen
(e g CEA), placental hormones (e g, chorionic gonadotropin), or isoenzymes feto-placental type (e g, placental alkaline phosphatase).
14. Ability to produce experimental animal tumors. This is the condition sine qua non that define the malignant transformation in vitro. I f the cells that are thought to
have been transformed do not product tumors in appropriate hosts animals, they can not be defined as "malignant". However, the failure to grow in an animal
model does not exclude the fact that they can be tumorigenic in a different kind of animal .
15. Ability to avoid host antitumor immune response.
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COLORECTAL CANCER
STATISTICS
In Europe colorectal cancer (cancer of the colon and rectum) is the
most common form of all the cancers.
Each year over 200,000 citizens in Europe die from colorectal
cancer (nearly two thirds of all cases diagnosed).
Colorectal cancer is the third most common cancer worldwide.
Yet this disease is preventable in most cases and highly treatable if
diagnosed in its very-early stage (pre-in situ stage ).
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COLORECTAL CANCER
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In colorectal cancer, to achieve the quantification of free
ribosomes there are, mainly, the following steps:
a) creation of a device (kit to collect stools which have thousands of mucosal cells
colonocytes - freed from the colonic and rectal mucosa, which are mixed with
and covering the stools voided);b) separation of the cells (colonocytes) from the stools;
c) homogenization and calibration of the colonocytes, through rupture of the
colonocytes, with certain routine techniques;d) isolation of the free ribosomes, though differential centrifugation;
e) labeling of the free ribosomes, utilizing fluorochromes (nanotechnology); and
f) counting labeled free ribosomes, utilizing flow cytometry or other method.
COLORECTAL CANCER (CRC)
PREPARATION AND COUNTING OF FREE
RIBOSOMES
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COLORECTAL CANCER (CRC)
ADVANTAGES OF RIBOGRAMA OVER OTHER
NONINVASIVE METHODS OF DETECTECCION OF CRC
The degree of public support will be quite high, given sum of
the following nice factors ":
01- No invasive act;02- No need for the patient, to handle their own feces during collection;
03- No fecal odor nuisance since the fecal collector is industrially designed
accordingly (see the design of the collector of feces in the following
images);
04- No bowel preparation is needed;
05- No need for cleansing enemas;
06- It is not necessary to use cathartics;
07- It is not necessary to shift the patient to a medical appointment or to a
medical center;
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08 - The patient does not have to change their way of life;
09 - The patient does not have to change the routine of their daily activities or
work;
10 - The patient has no need to change the habits and type of food;11 - The patient does not have to stop, or modify any medication;
12The method does not give false negatives or false positives, because the
test specification is limited to counting the ribosomes of colonocytes,
which are the targets to be studied in cases of colorectal cancer, or in
cases under malignization process, meaning that the test is 100%
specific, 100% reliable and universal, for all tissues, once the cells tostudy are separated.
COLORECTAL CANCER (CRC)
ADVANTAGES OF RIBOGRAMA OVER OTHER
NONINVASIVE METHODS OF DETECTECCION OF CRC
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RIBOGRAMA is an objective and quantitative method that provides an unique
service to HealthCare Providers worldwide. The method is qualified to deal within all
business sectors of the worldwide HealthCare, from the sole practice physician to the
largest chain of hospitals.
The advantages of RIBOGRAMA METHOD over other noninvasive tests of
cancer of the colon and rectum are the evident twelve "factors nice" that by themselves
provide a good patient compliance and a preference for determining the health
professionals, because there are no false positives or false negatives, since they areconsidering charges of ribosomes of cells isolated from the colorectal mucosa.
RIBOGRAMA
COLO RECTAL CANCER
FINAL REMARKS
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SUMMARYOF COLECTOR PARTS
SUPPORT PROTOTYPE OF STOOL COLLECTION
(COLLECTOR)
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SUMMARYOF COLECTOR PARTS
SUPPORT PROTOTYPE OF STOOL COLLECTION
(COLLECTOR)
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SUMMARYOF COLECTOR PARTS
SUPPORT PROTOTYPE OF STOOL COLLECTION
(COLLECTOR)
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SUMMARYOF COLECTOR PARTS
SUPPORT PROTOTYPE OF STOOL COLLECTION
(COLLECTOR)
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SUMMARYOF COLECTOR PARTS
SUPPORT PROTOTYPE OF STOOL COLLECTION
(COLLECTOR)
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The colorectal cancer represents one of the biggest incidences of human cancer in the western
type of life and cocked food.
This research project has strong potential in the following fields :
public health,
medico-hospitalar practice,
clinical and laboratorial research,
food industry,
veterinary,
oncologic research,
pharmaceutical industry and clinical trials research etc.,
The method has great medical and financial impact, firstly, by now, in the lowering significantly the
morbidity and mortality of the colorectal cancer disease, in the world, and, after, with other organs
and systems.
APLICATIONS
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Raise new questions, explore new possibilities, and
regard old problems from a new angle...
[Albert Einstein]
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The place where I was borned
and grown.
Oporto city - PORTUGAL